52 research outputs found
Apical localization of inositol 1,4,5-trisphosphate receptors is independent of extended synaptotagmins in hepatocytes.
Extended synaptotagmins (E-Syts) are a recently identified family of proteins that tether the endoplasmic reticulum (ER) to the plasma membrane (PM) in part by conferring regulation of cytosolic calcium (Ca2+) at these contact sites (Cell, 2013). However, the mechanism by which E-Syts link this tethering to Ca2+ signaling is unknown. Ca2+ waves in polarized epithelia are initiated by inositol 1,4,5-trisphosphate receptors (InsP3Rs), and these waves begin in the apical region because InsP3Rs are targeted to the ER adjacent to the apical membrane. In this study we investigated whether E-Syts are responsible for this targeting. Primary rat hepatocytes were used as a model system, because a single InsP3R isoform (InsP3R-II) is tethered to the peri-apical ER in these cells. Additionally, it has been established in hepatocytes that the apical localization of InsP3Rs is responsible for Ca2+ waves and secretion and is disrupted in disease states in which secretion is impaired. We found that rat hepatocytes express two of the three identified E-Syts (E-Syt1 and E-Syt2). Individual or simultaneous siRNA knockdown of these proteins did not alter InsP3R-II expression levels, apical localization or average InsP3R-II cluster size. Moreover, apical secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was not changed in cells lacking E-Syts but was reduced in cells in which cytosolic Ca2+ was buffered. These data provide evidence that E-Syts do not participate in the targeting of InsP3Rs to the apical region. Identifying tethers that bring InsP3Rs to the apical region remains an important question, since mis-targeting of InsP3Rs leads to impaired secretory activity
A novel physiological role for ARF1 in the formation of bidirectional tubules from the Golgi.
Capitalizing on CRISPR/Cas9 gene-editing techniques and super-resolution nanoscopy, we explore the role of the small GTPase ARF1 in mediating transport steps at the Golgi. Besides its well-established role in generating COPI vesicles, we find that ARF1 is also involved in the formation of long (∼3 µm), thin (∼110 nm diameter) tubular carriers. The anterograde and retrograde tubular carriers are both largely free of the classical Golgi coat proteins coatomer (COPI) and clathrin. Instead, they contain ARF1 along their entire length at a density estimated to be in the range of close packing. Experiments using a mutant form of ARF1 affecting GTP hydrolysis suggest that ARF1[GTP] is functionally required for the tubules to form. Dynamic confocal and stimulated emission depletion imaging shows that ARF1-rich tubular compartments fall into two distinct classes containing 1) anterograde cargoes and clathrin clusters or 2) retrograde cargoes and coatomer clusters
Evaluation of a commercial E(rns)-capture ELISA for detection of BVDV in routine diagnostic cattle serum samples
BACKGROUND: Bovine viral diarrhoea virus (BVDV) is an important pathogen in cattle. The ability of the virus to cross the placenta during early pregnancy can result in the birth of persistently infected (PI) calves. These calves shed the virus during their entire lifespan and are the key transmitters of infection. Consequently, identification (and subsequent removal) of PI animals is necessary to rapidly clear infected herds from the virus. The objective of this study was to evaluate the suitability of a commercial E(rns)-capture ELISA, in comparison to the indirect immunoperoxidase test (IPX), for routine diagnostic detection of BVDV within a control programme. In addition, the effect of passive immunity and heat-inactivation of the samples on the performance of the ELISA was studied. METHODS: In the process of virus clearance within the Swedish BVDV control programme, all calves born in infected herds are tested for virus and antibodies. From such samples, sent in for routine diagnostics to SVA, we selected 220 sera collected from 32 beef herds and 29 dairy herds. All sera were tested for BVDV antigen using the E(rns )ELISA, and the results were compared to the results from the IPX used within the routine diagnostics. RESULTS: All 130 samples categorized as virus negative by IPX were tested negative in the ELISA, and all 90 samples categorized as virus positive were tested positive, i.e. the relative sensitivity and specificity of the ELISA was 100% in relation to IPX, and the agreement between the tests was perfect. CONCLUSION: We can conclude that the E(rns )ELISA is a valid alternative that has several advantages compared to IPX. Our results clearly demonstrate that it performs well under Swedish conditions, and that its performance is comparable with the IPX test. It is highly sensitive and specific, can be used for testing of heat-inactivated samples, precolostral testing, and probably to detect PI animals at an earlier age than the IPX
The effect of childhood stunting and wasting on adolescent cardiovascular diseases risk and educational achievement in rural Uganda : a retrospective cohort study
Background: Little is known about the long-term effects of early childhood undernutrition on adolescent cardiovascular disease risk and educational performance in low-income countries. We examined this in a rural Ugandan population. Objective: To investigate if stunting or wasting among children aged 2-5 years is associated with cardiovascular disease risk or educational achievement during adolescence. Methods: We conducted analyses using data from a cohort of children followed from early childhood to adolescence. Weight and height were measured in 1999-2000 when the children were 2-5 years of age and repeated in 2004/2005 and 2011. We compared cardiovascular disease risk parameters (mean blood pressure, lipids, HbA1c) and schooling years achieved in 2011 among 1054 adolescents categorised into four groups: those who experienced stunting or wasting throughout follow-up; those who recovered from stunting or wasting; those who were normal but later became stunted or wasted; and those who never experienced stunting or wasting from childhood up to adolescence. We controlled for possible confounding using multiple generalised linear regression models along with Generalised Estimating Equations to account for clustering of children within households. Results: Wasting was negatively associated with systolic blood pressure (-7.90 95%CI [-14.52,-1.28], p = 0.02) and diastolic blood pressure (-3.92, 95%CI [-7.42, -0.38], p = 0.03). Stunting had borderline negative association with systolic blood pressure (-2.90, 95%CI [-6.41, 0.61] p = 0.10). Recovery from wasting was positively associated with diastolic blood pressure (1.93, 95%CI [0.11, 3.74] p = 0.04). Stunting or wasting was associated with fewer schooling years. Conclusion: Recovery from wasting rather than just an episode in early childhood is associated with a rise in blood pressure while educational achievement is compromised regardless of whether recovery from undernutrition happens. These findings are relevant to children exposed to undernutrition in low-income settings
Ultra-High Resolution 3D Imaging of Whole Cells.
Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80Â nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes
Variability and quasi-decadal changes in the methane budget overthe period 2000–2012
Following the recent Global Carbon Project (GCP)
synthesis of the decadal methane (CH4/ budget over 2000–
2012 (Saunois et al., 2016), we analyse here the same dataset
with a focus on quasi-decadal and inter-annual variability in
CH4 emissions. The GCP dataset integrates results from topdown
studies (exploiting atmospheric observations within an
atmospheric inverse-modelling framework) and bottom-up
models (including process-based models for estimating land
surface emissions and atmospheric chemistry), inventories of
anthropogenic emissions, and data-driven approaches.The annual global methane emissions from top-down studies,
which by construction match the observed methane
growth rate within their uncertainties, all show an increase in
total methane emissions over the period 2000–2012, but this
increase is not linear over the 13 years. Despite differences
between individual studies, the mean emission anomaly of the top-down ensemble shows no significant trend in total
methane emissions over the period 2000–2006, during
the plateau of atmospheric methane mole fractions, and also
over the period 2008–2012, during the renewed atmospheric
methane increase. However, the top-down ensemble mean
produces an emission shift between 2006 and 2008, leading
to 22 [16–32] Tg CH4 yr1 higher methane emissions
over the period 2008–2012 compared to 2002–2006. This
emission increase mostly originated from the tropics, with
a smaller contribution from mid-latitudes and no significant
change from boreal regions.
The regional contributions remain uncertain in top-down
studies. Tropical South America and South and East Asia
seem to contribute the most to the emission increase in the
tropics. However, these two regions have only limited atmospheric
measurements and remain therefore poorly constrained.
The sectorial partitioning of this emission increase between
the periods 2002–2006 and 2008–2012 differs from
one atmospheric inversion study to another. However, all topdown
studies suggest smaller changes in fossil fuel emissions
(from oil, gas, and coal industries) compared to the
mean of the bottom-up inventories included in this study.
This difference is partly driven by a smaller emission change
in China from the top-down studies compared to the estimate
in the Emission Database for Global Atmospheric Research
(EDGARv4.2) inventory, which should be revised to smaller
values in a near future. We apply isotopic signatures to the
emission changes estimated for individual studies based on
five emission sectors and find that for six individual top-down
studies (out of eight) the average isotopic signature of the
emission changes is not consistent with the observed change
in atmospheric 13CH4. However, the partitioning in emission
change derived from the ensemble mean is consistent with
this isotopic constraint. At the global scale, the top-down ensemble
mean suggests that the dominant contribution to the
resumed atmospheric CH4 growth after 2006 comes from microbial
sources (more from agriculture and waste sectors than
from natural wetlands), with an uncertain but smaller contribution
from fossil CH4 emissions. In addition, a decrease in
biomass burning emissions (in agreement with the biomass
burning emission databases) makes the balance of sources
consistent with atmospheric 13CH4 observations.
In most of the top-down studies included here, OH concentrations
are considered constant over the years (seasonal variations
but without any inter-annual variability). As a result,
the methane loss (in particular through OH oxidation) varies
mainly through the change in methane concentrations and not
its oxidants. For these reasons, changes in the methane loss
could not be properly investigated in this study, although it
may play a significant role in the recent atmospheric methane
changes as briefly discussed at the end of the paper.Published11135–111616A. Geochimica per l'ambienteJCR Journa
Land-locked mammalian Golgi reveals cargo transport between stable cisternae
The different composition of Golgi cisternae gave rise to two different models for intra-Golgi traffic: one where stable cisternae communicate via vesicles and another one where cisternae biochemically mature to ensure anterograde transport. Here, the authors provide evidence in support of the stable compartments model
Role of the Caenorhabditis elegans Multidrug Resistance Gene, mrp-4, in Gut Granule Differentiation
Caenorhabditis elegans gut granules are lysosome-related organelles with birefringent contents. mrp-4, which encodes an ATP-binding cassette (ABC) transporter homologous to mammalian multidrug resistance proteins, functions in the formation of gut granule birefringence. mrp-4(−) embryos show a delayed appearance of birefringent material in the gut granule but otherwise appear to form gut granules properly. mrp-4(+) activity is required for the extracellular mislocalization of birefringent material, body-length retraction, and NaCl sensitivity, phenotypes associated with defective gut granule biogenesis exhibited by embryos lacking the activity of GLO-1/Rab38, a putative GLO-1 guanine nucleotide exchange factor GLO-4, and the AP-3 complex. Multidrug resistance protein (MRP)-4 localizes to the gut granule membrane, consistent with it playing a direct role in the transport of molecules that compose and/or facilitate the formation of birefringent crystals within the gut granule. However, MRP-4 is also present in oocytes and early embryos, and our genetic analyses indicate that its site of action in the formation of birefringent material may not be limited to just the gut granule in embryos. In a search for genes that function similarly to mrp-4(+), we identified WHT-2, another ABC transporter that acts in parallel to MRP-4 for the formation of birefringent material in the gut granule
Function of the Caenorhabditis elegans ABC transporter PGP-2 in the biogenesis of a lysosome-related fat storage organelle
Caenorhabditis elegans gut granules are intestine specific lysosome-related organelles with birefringent and autofluorescent contents. We identified pgp-2, which encodes an ABC transporter, in screens for genes required for the proper formation of gut granules. pgp-2(-) embryos mislocalize birefringent material into the intestinal lumen and are lacking in acidified intestinal V-ATPase-containing compartments. Adults without pgp-2(+) function similarly lack organelles with gut granule characteristics. These cellular phenotypes indicate that pgp-2(-) animals are defective in gut granule biogenesis. Double mutant analysis suggests that pgp-2(+) functions in parallel with the AP-3 adaptor complex during gut granule formation. We find that pgp-2 is expressed in the intestine where it functions in gut granule biogenesis and that PGP-2 localizes to the gut granule membrane. These results support a direct role of an ABC transporter in regulating lysosome biogenesis. Previously, pgp-2(+) activity has been shown to be necessary for the accumulation of Nile Red-stained fat in C. elegans. We show that gut granules are sites of fat storage in C. elegans embryos and adults. Notably, levels of triacylglycerides are relatively normal in animals defective in the formation of gut granules. Our results provide an explanation for the loss of Nile Red-stained fat in pgp-2(-) animals as well as insight into the specialized function of this lysosome-related organelle
- …