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1.
Jacob J. Kennedy Ping Yan Lei Zhao Richard G. Ivey Uliana J. Voytovich Heather D. Moore Chenwei Lin Era L. Pogosova-Agadjanyan Derek L. Stirewalt Kerryn W. Reding Jeffrey R. Whiteaker Amanda G. Paulovich 《Molecular & cellular proteomics : MCP》2016,15(2):726-739
A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks.Cell signaling research is faced with the challenging task of interrogating increasingly large numbers of analytes in “systems biology” approaches, while maintaining the high standards of integrity and reproducibility traditionally associated with the scientific approach. For example, studies interrogating complex systems, such as protein signaling networks, require quantification technologies capable of sensitive, specific, multiplexable, and reproducible application. However, recent reports have highlighted alarmingly high rates of irreproducibility in fundamental biological and pre-clinical studies (1, 2), as well as poor performance of affinity reagents used in traditional proteomic assay and detection platforms (3, 4). There is an imminent need for high quality assays, including highly characterized standards and detailed documentation of processes and procedures (5). To improve the translation of cell signaling discoveries into clinical application, we need reproducible and transferable technologies that enable higher throughput quantification of protein phosphorylation.Signaling dynamics through post-translational modifications (e.g. phosphorylation) are predominantly measured by Western blotting. Although this technique has led to many discoveries and is the de facto “gold standard,” it suffers from many drawbacks. Western blotting is a low throughput approach applied to individual analytes (i.e. no multiplexing) and is susceptible to erroneous interpretation when applied quantitatively (6). Alternative immunoassay platforms have emerged (e.g. immunohistochemistry, ELISA, mass cytometry, and bead-based or planar arrays), but suffer from similar limitations, namely specificity issues (because of cross-reactivity of antibodies), poor standardization, and difficulties in multiplexing.One alternative for quantifying phosphorylation is targeted, multiple reaction monitoring (MRM)1 MS, a widely deployed technique in clinical laboratories for quantification of small molecules (7, 8). MRM is now also well established for precise and specific quantification of endogenous, proteotypic peptides relative to spiked-in stable isotope-labeled internal standards (9–11), and MRM can be applied to phosphopeptides (12–18). MRM assays can be run at high multiplex levels (19–21) and can be standardized to be highly reproducible across laboratories (22–24), even on an international stage (25). Because phosphorylation typically occurs at sub-stoichiometric levels and because phosphopeptides must compete for ionization with more abundant peptides, mass spectrometry-based analysis of phosphorylation requires an analyte enrichment step. Immuno-affinity enrichment approaches using anti-phospho-tyrosine antibodies (26) or panels of antibodies targeting signaling nodes (27) have been implemented with shotgun mass spectrometry. Although anti-peptide antibodies can also be used to enrich individual phosphopeptides upstream of MRM (28), the generation of these reagents is time-consuming and costly, limiting widespread uptake.Phosphopeptide enrichment based on metal affinity chromatography has recently matured into a reproducible approach (29). Immobilized metal affinity chromatography (IMAC) is widely used in discovery phosphoproteomic studies to enrich phosphopeptides upstream of shotgun-based mass spectrometry (30, 31). We hypothesized that a subset of the cellular phosphoproteome with favorable binding characteristics to the IMAC resin might be reproducibly recovered for quantification when coupled with quantitative MRM mass spectrometry, enabling robust IMAC-MRM assays without the need for an antibody.In this report, we: (1) demonstrate the feasibility of generating analytically robust, multiplex IMAC-MRM assays for quantifying cellular phospho-signaling, (2) present a semi-automated, 96-well format magnetic bead-based protocol for IMAC enrichment, (3) provide a catalogue of phosphopeptides that are highly amenable to IMAC-MRM quantification, and (4) make publicly available standard operating protocols (SOP) and fit-for-purpose analytical validation data for IMAC-MRM assays targeting 107 phospho-analytes, providing a community resource for study of the DNA damage response. The data suggest that the IMAC-MRM approach is generally applicable to signaling pathways, enabling wider interrogation of signaling networks. 相似文献
2.
Slack KE Delsuc F McLenachan PA Arnason U Penny D 《Molecular phylogenetics and evolution》2007,42(1):1-13
Incomplete taxon sampling has been a major problem in resolving the early divergences in birds. Five new mitochondrial genomes are reported here (brush-turkey, lyrebird, suboscine flycatcher, turkey vulture, and a gull) and three break up long branches that tended to attract the distant reptilian outgroup. These long branches were to galliforms, and to oscine and suboscine passeriformes. Breaking these long branches leaves the root, as inferred by maximum likelihood and Bayesian phylogenetic analyses, between paleognaths and neognaths. This means that morphological, nuclear, and mitochondrial data are now in agreement on the position of the root of the avian tree and we can, move on to other questions. An overview is then given of the deepest divisions in the mitogenomic tree inferred from complete mitochondrial genomes. The strict monophyly of both the galloanseres and the passerines is strongly supported, leaving the deep six-way split within Neoaves as the next major question for which resolution is still lacking. Incomplete taxon sampling was also a problem for Neoaves, and although some resolution is now available there are still problems because current phylogenetic methods still fail to account for real features of DNA sequence evolution. 相似文献
3.
Germana Bancone Nongnud Chowwiwat Raweewan Somsakchaicharoen Lalita Poodpanya Paw Khu Moo Gornpan Gornsawun Ladda Kajeechiwa May Myo Thwin Santisuk Rakthinthong Suphak Nosten Suradet Thinraow Slight Naw Nyo Clare L. Ling Jacher Wiladphaingern Naw Lily Kiricharoen Kerryn A. Moore Nicholas J. White Francois Nosten 《PloS one》2016,11(3)
Background
Primaquine is the only drug consistently effective against mature gametocytes of Plasmodium falciparum. The transmission blocking dose of primaquine previously recommended was 0.75mg/kg (adult dose 45mg) but its deployment was limited because of concerns over haemolytic effects in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. G6PD deficiency is an inherited X-linked enzymatic defect that affects an estimated 400 million people around the world with high frequencies (15–20%) in populations living in malarious areas. To reduce transmission in low transmission settings and facilitate elimination of P. falciparum, the World Health Organization now recommends adding a single dose of 0.25mg/kg (adult dose 15mg) to Artemisinin-based Combination Therapies (ACTs) without G6PD testing. Direct evidence of the safety of this low dose is lacking. Adverse events and haemoglobin variations after this treatment were assessed in both G6PD normal and deficient subjects in the context of targeted malaria elimination in a malaria endemic area on the North-Western Myanmar-Thailand border where prevalence of G6PD deficiency (Mahidol variant) approximates 15%.Methods and Findings
The tolerability and safety of primaquine (single dose 0.25 mg base/kg) combined with dihydroartemisinin-piperaquine (DHA-PPQ) given three times at monthly intervals was assessed in 819 subjects. Haemoglobin concentrations were estimated over the six months preceding the ACT + primaquine rounds of mass drug administration. G6PD deficiency was assessed with a phenotypic test and genotyping was performed in male subjects with deficient phenotypes and in all females. Fractional haemoglobin changes in relation to G6PD phenotype and genotype and primaquine round were assessed using linear mixed-effects models. No adverse events related to primaquine were reported during the trial. Mean fractional haemoglobin changes after each primaquine treatment in G6PD deficient subjects (-5.0%, -4.2% and -4.7%) were greater than in G6PD normal subjects (0.3%, -0.8 and -1.7%) but were clinically insignificant. Fractional drops in haemoglobin concentration larger than 25% following single dose primaquine were observed in 1.8% of the population but were asymptomatic.Conclusions
The single low dose (0.25mg/kg) of primaquine is clinically well tolerated and can be used safely without prior G6PD testing in populations with high prevalence of G6PD deficiency. The present evidence supports a broader use of low dose primaquine without G6PD testing for the treatment and elimination of falciparum malaria.Trial Registration
ClinicalTrials.gov NCT01872702 相似文献4.
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Guy Balme Matt Rogan Lisa Thomas Ross Pitman Gareth Mann Gareth Whittington-Jones Neil Midlane Mark Broodryk Kerryn Broodryk Michelle Campbell Marc Alkema Dave Wright Luke Hunter 《Population Ecology》2019,61(3):256-267
Human impact is near pervasive across the planet and studies of wildlife populations free of anthropogenic mortality are increasingly scarce. This is particularly true for large carnivores that often compete with and, in turn, are killed by humans. Accordingly, the densities at which carnivore populations occur naturally, and their role in shaping and/or being shaped by natural processes, are frequently unknown. We undertook a camera-trap survey in the Sabi Sand Game Reserve (SSGR), South Africa, to examine the density, structure and spatio-temporal patterns of a leopard Panthera pardus population largely unaffected by anthropogenic mortality. Estimated population density based on spatial capture–recapture models was 11.8 ± 2.6 leopards/100 km2. This is likely close to the upper density limit attainable by leopards, and can be attributed to high levels of protection (particularly, an absence of detrimental edge effects) and optimal habitat (in terms of prey availability and cover for hunting) within the SSGR. Although our spatio-temporal analyses indicated that leopard space use was modulated primarily by “bottom-up” forces, the population appeared to be self-regulating and at a threshold that is unlikely to change, irrespective of increases in prey abundance. Our study provides unique insight into a naturally-functioning carnivore population at its ecological carrying capacity. Such insight can potentially be used to assess the health of other leopard populations, inform conservation targets, and anticipate the outcomes of population recovery attempts. 相似文献
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Wlodek ME Westcott KT O'Dowd R Serruto A Wassef L Moritz KM Moseley JM 《American journal of physiology. Regulatory, integrative and comparative physiology》2005,288(6):R1620-R1627
During pregnancy, parathyroid hormone-related protein (PTHrP) is one of many growth factors that play important roles to promote fetal growth and development, including stimulation of placental calcium transport. Angiotensin II, acting through the AT(1a) receptor, is also known to promote placental growth. We examined the effects of bilateral uterine artery and vein ligation (restriction), which mimics placental insufficiency in humans, on growth, intrauterine PTHrP, placental AT(1a), and pup calcium. Growth restriction was surgically induced on day 18 of pregnancy in Wistar-Kyoto female rats by uterine vessel ligation. Uteroplacental insufficiency reduced fetal body weight by 15% and litter size (P < 0.001) compared with the control rats with no effect on placental weight or amniotic fluid volume. Uteroplacental insufficiency reduced placental PTHrP content by 46%, with increases in PTHrP (by 2.6-fold), parathyroid hormone (PTH)/PTHrP receptor (by 11.6-fold), and AT(1a) (by 1.7-fold) relative mRNA in placenta following restriction compared with results in control (P < 0.05). There were no alterations in uterine PTHrP and PTH/PTHrP receptor mRNA expression. Maternal and fetal plasma PTHrP and calcium concentrations were unchanged. Although fetal total body calcium was not altered, placental restriction altered perinatal calcium homeostasis, as evidenced by lower pup total body calcium after birth (P < 0.05). The increased uterine and amniotic fluid PTHrP (P < 0.05) may be an attempt to compensate for the induced impaired placental function. The present study demonstrates that uteroplacental insufficiency alters intrauterine PTHrP, placental AT(1a) expression, and perinatal calcium in association with a reduction in fetal growth. Uteroplacental insufficiency may provide an important model for exploring the early origins of adult diseases. 相似文献
8.
Kerryn?A.?Chia "mailto:chia@wn.com.au " title= "chia@wn.com.au " itemprop= "email " data-track= "click " data-track-action= "Email author " data-track-label= " ">Email author "http://orcid.org/--- " itemprop= "url " title= "View OrcID profile " target= "_blank " rel= "noopener " data-track= "click " data-track-action= "OrcID " data-track-label= " ">View author&#;s OrcID profile John?M.?Koch Rohan?Sadler Shane?R.?Turner 《Ecological Research》2016,31(5):627-638
Persoonia longifolia is a common mid-storey species that is difficult to return to post-mining environments. This study aimed to quantify in situ emergence of P. longifolia seeds on restored areas, investigate seed cueing prior to use in restoration and assess different tree guards for increasing seedling survival and health. Initial investigations found that <1 % of seeds buried or scattered on restored areas produced seedlings. However, if seeds were cued through burial in surrounding forest, retrieved and sown on restored areas, seedling emergence increased to 24 %. Significantly more seeds emerged as seedlings when buried (14.6 %) compared to those scattered on the soil surface (2.7 %). There was no significant difference in survival between seedlings planted at 2-3 weeks of age compared with those planted at 12 months of age after 20 months in situ growth. Additionally, those seedlings planted when younger were significantly taller (29.0 ± 2.9 cm) than those that were planted at 12 months of age (4.7 ± 0.3 cm). Use of “onion bag” guards improved survival from 58.1 ± 4.0 % (no guard) to 70.8 ± 3.4 % with an onion bag guard. The use of shade cloth guards did not significantly improve survival, however plant height did increase substantially after 32 months growth (22 cm compared with 7.2 cm for no guard). These data demonstrate that consideration needs to be given to specific species requirements to improve seedling emergence and survival when attempting to return difficult to germinate species to the post-mining environment. 相似文献
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In arid/semi‐arid environments, where rainfall is seasonal, highly variable and significantly less than the evaporation rate, groundwater discharge can be a major component of the water and salt balance of a wetland, and hence a major determinant of wetland ecology. Under natural conditions, wetlands in arid/semi‐arid zones occasionally experience periods of higher salinity as a consequence of the high evaporative conditions and the variability of inflows which provide dilution and flushing of the stored salt. However, due to the impacts of human population pressure and the associated changes in land use, surface water regulation, and water resource depletion, wetlands in arid/semi‐arid environments are now often experiencing extended periods of high salinity. This article reviews the current knowledge of the role that groundwater–surface water (GW–SW) interactions play in the ecology of arid/semi‐arid wetlands. The key findings of the review are as follows:
- 1. GW–SW interactions in wetlands are highly dynamic, both temporally and spatially. Groundwater that is low in salinity has a beneficial impact on wetland ecology which can be diminished in dry periods when groundwater levels, and hence, inflows to wetlands are reduced or even cease. Conversely, if groundwater is saline, and inflows increase due to raised groundwater levels caused by factors such as land use change and river regulation, then this may have a detrimental impact on the ecology of a wetland and its surrounding areas.
- 2. GW–SW interactions in wetlands are mostly controlled by factors such as differences in head between the wetland surface water and groundwater, the local geomorphology of the wetland (in particular, the texture and chemistry of the wetland bed and banks), and the wetland and groundwater flow geometry. The GW–SW regime can be broadly classified into three types of flow regimes: (i) recharge—wetland loses surface water to the underlying aquifer; (ii) discharge—wetland gains water from the underlying aquifer; or (iii) flow‐through—wetland gains water from the groundwater in some locations and loses it in others. However, it is important to note that individual wetlands may temporally change from one type to another depending on how the surface water levels in the wetland and the underlying groundwater levels change over time in response to climate, land use, and management.
- 3. The salinity in wetlands of arid/semi‐arid environments will vary naturally due to high evaporative conditions, sporadic rainfall, groundwater inflows, and freshening after rains or floods. However, wetlands are often at particular risk of secondary salinity because their generally lower elevation in the landscape exposes them to increased saline groundwater inflows caused by rising water tables. Terminal wetlands are potentially at higher risk than flow‐through systems as there is no salt removal mechanism.
- 4. Secondary salinity can impact on wetland biota through changes in both salinity and water regime, which result from the hydrological and hydrogeological changes associated with secondary salinity. Whilst there have been some detailed studies of these interactions for some Australian riparian tree species, the combined effects on aquatic biodiversity are only just beginning to be elucidated, and are therefore, a future research need.
- 5. Rainfall/flow‐pulses, which are a well‐recognized control on ecological function in arid/semi‐arid areas, also play an important, though indirect, role through their impact on wetland salinity. Freshwater pulses can be the primary means by which salt stored in both the water column and the underlying sediments are flushed from wetlands. Conversely, increased runoff is also a commonly observed consequence of secondary salinity, and so, wetlands can experience increased surface water inflows that are higher in salinity than under natural conditions. Moreover, changes in rainfall/flow‐pulse regimes can have a significant impact on wetland GW–SW interactions. It is possible that in some instances groundwater inflow to a wetland may become so heavy that it could become a major component of the water balance, and hence, mask the role of natural pulsing regimes. However, if the groundwater is low in salinity, this may provide an ecological benefit in arid/semi‐arid areas by assisting in maintaining water in wetlands that become aquatic refugia between flow‐pulses.
- 6. There has been almost no modelling of GW–SW interactions in arid/semi‐arid wetlands with respect to water fluxes, let alone salinity or ecology. There is a clear need to develop modelling capabilities for the movement of salt to, from, and within wetlands to provide temporal predictions of wetland salinity which can be used to assess ecosystem outcomes.
- 7. There has been a concerted effort in Australia to collect and collate data on the salinity tolerance/sensitivity of freshwater aquatic biota and riparian vegetation. There are many shortcomings and knowledge gaps in these data, a fact recognized by many of the authors of this work. Particularly notable is that there is very little time‐series data, which is a serious issue because wetland salinities are often highly temporally variable. There is also a concern that many of the data are from very controlled laboratory experiments, which may not represent the highly variable and unpredictable conditions experienced in the field. In light of these, and many other shortcomings identified, our view is that the data currently available are a useful guide but must be used with some caution. Copyright © 2008 John Wiley & Sons, Ltd.