Production, characterisation and use of fluorescent markers in the study of plant nuclear envelope dynamics

The nuclear envelope (NE) is one of the least studied membranes in plant cells. Genes encoding NE protein homologues are absent from currently available sequenced plant genomes. To produce a specific marker for the plant NE and in view of previous positive results with heterologously expressed proteins in plant cells, mammalian NE proteins were considered in order to find a marker for the plant NE. Green fluorescent protein (GFP) was chosen to label the protein of interest as it provides a non-invasive method of monitoring protein location and movement in vivo. The lamin B receptor (LBR) is the most extensively studied of the mammalian inner NE (INE) proteins. It is an INE protein that binds lam in B, chromatin and chromatin-associated proteins. A LBR-GFPs construct was produced and placed in a plant expression vector. Transient expression of the LBR-GFPs protein in tobacco leaf epidermal cells showed labelling of the NE, with minimal labelling of the endoplasmic reticulum (ER). The construct was used to produce stably transformed tobacco plants and tobacco BY -2 cells. NE labelling was observed in the majority of tissues stably expressing the protein, with NE location confirmed by electron microscopy. In BY -2 cells the construct showed NE location during interphase, with co-localisation with an ER marker, sporamin signal peptide YFP-HDEL (sp Y~P:"HDEL) during mitosis. Similar labelling of the ER with NE proteins is seen in mammalian cells during division. This, in combination with the targeting and retention of LBR-GFPs at the plant NE suggests a conservation of mechanisms 'for INE targeting and retention in plant and animal NEs. Preliminary evaluation of the nature of LBR retention at the NE was conducted using a set of LBR-GFPs mutants. Labelling of the NE was perturbed with some of the mutants, indicating that similar domains contribute to LBR retention in plant and ,animal cells

In cultured cells the baculovirus P10 protein forms two independent intracellular structures that play separate roles in occlusion body maturation and their release by nuclear disintegration

P10 is a small, abundant baculovirus protein that accumulates to high levels in the very late stages of the infection cycle. It is associated with a number of intracellular structures and implicated in diverse processes from occlusion body maturation to nuclear stability and lysis. However, studies have also shown that it is non-essential for virus replication, at least in cell culture. Here, we describe the use of serial block-face scanning electron microscopy to achieve high-resolution 3D characterisation of P10 structures within Trichoplusia ni TN-368 cells infected with Autographa californica multiple nucleopolyhedrovirus. This has enabled unparalleled visualisation of P10 and determined the independent formation of dynamic perinuclear and nuclear vermiform fibrous structures. Our 3D data confirm the sequence of ultrastructural changes that create a perinuclear cage from thin angular fibrils within the cytoplasm. Over the course of infection in cultured cells, the cage remodels to form a large polarised P10 mass and we suggest that these changes are critical for nuclear lysis to release occlusion bodies. In contrast, nuclear P10 forms a discrete vermiform structure that was observed in close spatial association with both electron dense spacers and occlusion bodies; supporting a previously suggested role for P10 and electron dense spacers in the maturation of occlusion bodies. We also demonstrate that P10 hyper-expression is critical for function. Decreasing levels of p10 expression, achieved by manipulation of promoter length, correlated with reduced P10 production, a lack of formation of P10 structures and a concomitant decrease in nuclear lysis

The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies

Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology

Regulatory T Cell Responses in Participants with Type 1 Diabetes after a Single Dose of Interleukin-2: A Non-Randomised, Open Label, Adaptive Dose-Finding Trial

BACKGROUND: Interleukin-2 (IL-2) has an essential role in the expansion and function of CD4+ regulatory T cells (Tregs). Tregs reduce tissue damage by limiting the immune response following infection and regulate autoreactive CD4+ effector T cells (Teffs) to prevent autoimmune diseases, such as type 1 diabetes (T1D). Genetic susceptibility to T1D causes alterations in the IL-2 pathway, a finding that supports Tregs as a cellular therapeutic target. Aldesleukin (Proleukin; recombinant human IL-2), which is administered at high doses to activate the immune system in cancer immunotherapy, is now being repositioned to treat inflammatory and autoimmune disorders at lower doses by targeting Tregs. METHODS AND FINDINGS: To define the aldesleukin dose response for Tregs and to find doses that increase Tregs physiologically for treatment of T1D, a statistical and systematic approach was taken by analysing the pharmacokinetics and pharmacodynamics of single doses of subcutaneous aldesleukin in the Adaptive Study of IL-2 Dose on Regulatory T Cells in Type 1 Diabetes (DILT1D), a single centre, non-randomised, open label, adaptive dose-finding trial with 40 adult participants with recently diagnosed T1D. The primary endpoint was the maximum percentage increase in Tregs (defined as CD3+CD4+CD25highCD127low) from the baseline frequency in each participant measured over the 7 d following treatment. There was an initial learning phase with five pairs of participants, each pair receiving one of five pre-assigned single doses from 0.04 × 106 to 1.5 × 106 IU/m2, in order to model the dose-response curve. Results from each participant were then incorporated into interim statistical modelling to target the two doses most likely to induce 10% and 20% increases in Treg frequencies. Primary analysis of the evaluable population (n = 39) found that the optimal doses of aldesleukin to induce 10% and 20% increases in Tregs were 0.101 × 106 IU/m2 (standard error [SE] = 0.078, 95% CI = -0.052, 0.254) and 0.497 × 106 IU/m2 (SE = 0.092, 95% CI = 0.316, 0.678), respectively. On analysis of secondary outcomes, using a highly sensitive IL-2 assay, the observed plasma concentrations of the drug at 90 min exceeded the hypothetical Treg-specific therapeutic window determined in vitro (0.015-0.24 IU/ml), even at the lowest doses (0.040 × 106 and 0.045 × 106 IU/m2) administered. A rapid decrease in Treg frequency in the circulation was observed at 90 min and at day 1, which was dose dependent (mean decrease 11.6%, SE = 2.3%, range 10.0%-48.2%, n = 37), rebounding at day 2 and increasing to frequencies above baseline over 7 d. Teffs, natural killer cells, and eosinophils also responded, with their frequencies rapidly and dose-dependently decreased in the blood, then returning to, or exceeding, pretreatment levels. Furthermore, there was a dose-dependent down modulation of one of the two signalling subunits of the IL-2 receptor, the β chain (CD122) (mean decrease = 58.0%, SE = 2.8%, range 9.8%-85.5%, n = 33), on Tregs and a reduction in their sensitivity to aldesleukin at 90 min and day 1 and 2 post-treatment. Due to blood volume requirements as well as ethical and practical considerations, the study was limited to adults and to analysis of peripheral blood only. CONCLUSIONS: The DILT1D trial results, most notably the early altered trafficking and desensitisation of Tregs induced by a single ultra-low dose of aldesleukin that resolves within 2-3 d, inform the design of the next trial to determine a repeat dosing regimen aimed at establishing a steady-state Treg frequency increase of 20%-50%, with the eventual goal of preventing T1D. TRIAL REGISTRATION: ISRCTN Registry ISRCTN27852285; ClinicalTrials.gov NCT01827735.This is the final version of the article. It first appeared from the Public Library of Science via http://dx.doi.org/10.1371/journal.pmed.100213

Clam Terrace Rock Walls: The Ecology and Social Significance of Monumental Places

When we think of the monumental works of the indigenous peoples of the Northwest Coast, we typically think of long houses, canoes, and totem poles. But the First Peoples are also monumental movers of stone in the making of resource sites like clam terraces and root gardens. Clam terraces are special places that enhance beaches for clam production, and are used to harvest not only clams but an entire suite of algae and animals. While many studies have focused on the ability of clam gardens to enhance clam productivity, few have examined the role of the rock wall itself. These stories describe the monumental work of Indigenous peoples of the Northwest Coast to create these rock wall structures, indelibly shaping land and seascapes with their creation and inscribing the physical world with visible records of familial connections to places, while significantly managing food resources. In other words, the moving of stone at this monumental scale entangles the production of key foods with the making histories and places. These stories highlight the importance of these features in shaping our seascapes as well as our ecosystems. By creating new habitat, rock walls change intertidal ecological communities and alter the availability of non-clam food species, bringing foods like crab, urchin, seaweeds, and octopus into the garden and onto the plate