Rainbow trout (Oncorhynchus mykiss) is a salmonid species with a complex life-history. Wild populations are naturally divided into freshwater residents and sea-run migrants. Migrants undergo an ...energy-demanding adaptation for life in seawater, known as smoltification, while freshwater residents display these changes in an attenuated magnitude and rate. Despite this, in seawater rainbow trout farming all fish are transferred to seawater. Under these circumstances, weeks after seawater transfer, a significant portion of the fish die (around 10%) or experience growth stunting (GS; around 10%), which represents an important profitability and welfare issue. The underlying causes leading to GS in seawater-transferred rainbow trout remain unknown. In this study, we aimed at characterising the GS phenotype in seawater-transferred rainbow trout using untargeted and targeted approaches. To this end, the liver proteome (LC-MS/MS) and lipidome (LC-MS) of GS and fast-growing phenotypes were profiled to identify molecules and processes that are characteristic of the GS phenotype. Moreover, the transcription, abundance or activity of key proteins and hormones related to osmoregulation (Gill Na+, K + -ATPase activity), growth (plasma IGF-I, and liver igf1, igfbp1b, ghr1 and ctsl) and stress (plasma cortisol) were measured using targeted approaches. No differences in Gill Na+, K + -ATPase activity and plasma cortisol were detected between the two groups. However, a significant downregulation in plasma IGF-I and liver igf1 transcription pointed at this growth factor as an important pathomechanism for GS. Changes in the liver proteome revealed reactive-oxygen-species-mediated endoplasmic reticulum stress as a key mechanism underlying the GS phenotype. From the lipidomic analysis, key observations include a reduction in triacylglycerols and elevated amounts of cardiolipins, a characteristic lipid class associated with oxidative stress, in GS phenotype. While the triggers to the activation of endoplasmic reticulum stress are still unknown, data from this study point towards a nutritional deficiency as an underlying driver of this phenotype.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Parental investment in Arapaima gigas includes nest building and guarding, followed by a care provision when a cephalic fluid is released from the parents' head to the offspring. This fluid has ...presumably important functions for the offspring but so far its composition has not been characterised. In this study the proteome and peptidome of the cephalic secretion was studied in parental and non-parental fish using capillary electrophoresis coupled to mass spectrometry (CE-MS) and GeLC-MS/MS analyses. Multiple comparisons revealed 28 peptides were significantly different between males and parental males (PC-males), 126 between females and parental females (PC-females), 51 between males and females and 9 between PC-males and PC-females. Identification revealed peptides were produced in the inner ear (pcdh15b), eyes (tetraspanin and ppp2r3a), central nervous system (otud4, ribeye a, tjp1b and syn1) among others. A total of 422 proteins were also identified and gene ontology analysis revealed 28 secreted extracellular proteins. From these, 2 hormones (prolactin and stanniocalcin) and 12 proteins associated to immunological processes (serotransferrin, α-1-antitrypsin homolog, apolipoprotein A-I, and others) were identified. This study provides novel biochemical data on the lateral line fluid which will enable future hypotheses-driven experiments to better understand the physiological roles of the lateral line in chemical communication.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
SOCCs (store-operated Ca(2+) channels) are highly selective ion channels that are activated upon release of Ca(2+) from intracellular stores to regulate a multitude of diverse cellular functions. It ...was reported previously that Golli-BG21, a member of the MBP (myelin basic protein) family of proteins, regulates SOCE (store-operated Ca(2+) entry) in T-cells and oligodendrocyte precursor cells, but the underlying mechanism for this regulation is unknown. In the present study we have discovered that Golli can directly interact with the ER (endoplasmic reticulum) Ca(2+)-sensing protein STIM1 (stromal interaction molecule 1). Golli interacts with the C-terminal domain of STIM1 in both in vitro and in vivo binding assays and this interaction may be modulated by the intracellular Ca(2+) concentration. Golli also co-localizes with full-length STIM1 and Orai1 complexes in HeLa cells following Ca(2+) store depletion. Overexpression of Golli reduces SOCE in HeLa cells, but this inhibition is overcome by overexpressing STIM1. We therefore suggest that Golli binds to STIM1-Orai1 complexes to negatively regulate the activity of SOCCs.
Atherothrombotic disease is a well-recognized complication of diabetes and is a major contributor to the high morbidity and mortality associated with diabetes. Although there is substantial evidence ...linking diabetes with cardiovascular disease, the specific effect of hyper- (or hypo-) glycaemia is less well understood. The present review focuses on the impact that glycaemic dysregulation has on respiratory function and ROS (reactive oxygen species) generation in the endothelial cells that are critical in preventing several key steps in the atherothrombotic process. Endothelial cells are particularly susceptible to ROS-mediated dysfunction not only because of reduced cell viability and increased senescence, but also because one of the major endothelium-derived factors that help to protect against atherosclerosis, nitric oxide, is rapidly deactivated by superoxide radicals.
An important area of proteomics involves the need for quantification, whether relative or absolute. Many methods now exist for relative quantification, but to support biomarker proteomics and systems ...biology, absolute quantification rather than relative quantification is required. Absolute quantification usually involves the concomitant mass spectrometric determination of signature proteotypic peptides and stable isotope-labeled analogs. However, the availability of standard labeled signature peptides in accurately known amounts is a limitation to the widespread adoption of this approach. We describe the design and synthesis of artificial QconCAT proteins that are concatamers of tryptic peptides for several proteins. This protocol details the methods for the design, expression, labeling, purification, characterization and use of the QconCATs in the absolute quantification of complex protein mixtures. The total time required to complete this protocol (from the receipt of the QconCAT expression plasmid to the absolute quantification of the set of proteins encoded by the QconCAT protein in an analyte sample) is approximately 29 d.
Acylated amino acids function as important components of the cellular membrane in some bacteria. Biosynthesis is initiated by the
-acylation of the amino acid, and this is followed by subsequent
...-acylation of the acylated molecule, resulting in the production of the mature diacylated amino acid lipid. In this study, we use both genetics and liquid chromatography-mass spectrometry (LC-MS) to characterize the biosynthesis and function of a diacylated glycine lipid (GL) species produced in
We, and others, have previously reported the identification of a gene, named
in this study, that encodes an
-acyltransferase activity responsible for the production of a monoacylated glycine called
-acyl-3-hydroxy-palmitoyl glycine (or commendamide). In all of the
genomes sequenced so far, the
gene is located immediately downstream from a gene, named
, that is also predicted to encode a protein with acyltransferase activity. We use LC-MS to show that the coexpression of
and
results in the production of GL in
We constructed a deletion mutant of the
gene in
, and we confirm that
is required for the production of GL in
Moreover, we show that
is important for the ability of
to adapt to stress and colonize the mammalian gut. Therefore, this report describes the genetic requirements for the biosynthesis of GL, a diacylated amino acid species that contributes to fitness in the human gut bacterium
The gut microbiome has an important role in both health and disease of the host. The mammalian gut microbiome is often dominated by bacteria from the
, an order that includes
and
In this study, we have identified an acylated amino acid, called glycine lipid, produced by
, a beneficial bacterium originally isolated from the human gut. In addition to identifying the genes required for the production of glycine lipids, we show that glycine lipids have an important role during the adaptation of
to a number of environmental stresses, including exposure to either bile or air. We also show that glycine lipids are important for the normal colonization of the murine gut by
This work identifies glycine lipids as an important fitness determinant in
and therefore increases our understanding of the molecular mechanisms underpinning colonization of the mammalian gut by beneficial bacteria.
Mutations in glucocerebrosidase (
GBA
) are the most prevalent genetic risk factor for Lewy body disorders (LBD)—collectively Parkinson’s disease, Parkinson’s disease dementia and dementia with Lewy ...bodies. Despite this genetic association, it remains unclear how
GBA
mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and α-synuclein levels in brain tissue from LBD and controls, with and without
GBA
mutations. We show that LBD is characterised by altered sphingolipid metabolism with prominent elevation of ceramide species, regardless of
GBA
mutations. Since extracellular vesicles (EV) could be involved in LBD pathogenesis by spreading disease-linked lipids and proteins, we investigated EV derived from
post-mortem
cerebrospinal fluid (CSF) and brain tissue from
GBA
mutation carriers and non-carriers. EV purified from LBD CSF and frontal cortex were heavily loaded with ceramides and neurodegeneration-linked proteins including alpha-synuclein and tau. Our in vitro studies demonstrate that LBD EV constitute a “pathological package” capable of inducing aggregation of wild-type alpha-synuclein, mediated through a combination of alpha-synuclein–ceramide interaction and the presence of pathological forms of alpha-synuclein. Together, our findings indicate that abnormalities in ceramide metabolism are a feature of LBD, constituting a promising source of biomarkers, and that
GBA
mutations likely accelerate the pathological process occurring in sporadic LBD through endolysosomal deficiency.
Nitric oxide (NO) storage and release measurements have been recorded for Ni-doped CPO-27 (Mg) and CPO-27 (Zn), and the biological effect of the released NO was assessed in porcine coronary artery ...relaxation tests. The results indicate that the doping strategy leads to increased levels of NO storage and delivery compared to the parent materials and that the NO dosage and biological response can be tuned
this approach to suit the requirements of particular applications.
Macrophages play a central role in inflammation by phagocytosing invading pathogens, apoptotic cells and debris, as well as mediating repair of tissues damaged by trauma. In order to do this, these ...dynamic cells generate a variety of inflammatory mediators including eicosanoids such as prostaglandins, leukotrienes and hydroxyeicosatraenoic acids (HETEs) that are formed through the cyclooxygenase, lipoxygenase and cytochrome P450 pathways. The ability to examine the effects of eicosanoid production at the protein level is therefore critical to understanding the mechanisms associated with macrophage activation.
This study presents a stable isotope labelling with amino acids in cell culture (SILAC) -based proteomics strategy to quantify the changes in macrophage protein abundance following inflammatory stimulation with Kdo2-lipid A and ATP, with a focus on eicosanoid metabolism and regulation. Detailed gene ontology analysis, at the protein level, revealed several key pathways with a decrease in expression in response to macrophage activation, which included a promotion of macrophage polarisation and dynamic changes to energy requirements, transcription and translation. These findings suggest that, whilst there is evidence for the induction of a pro-inflammatory response in the form of prostaglandin secretion, there is also metabolic reprogramming along with a change in cell polarisation towards a reduced pro-inflammatory phenotype.
Advanced quantitative proteomics in conjunction with functional pathway network analysis is a useful tool to investigate the molecular pathways involved in inflammation.
What's already known about this topic?
Current professional guidelines regarding the use of chromosomal microarray analysis (CMA) versus karyotyping in prenatal diagnosis support CMA over karyotype ...only when fetal structural abnormalities are present.
Examination of the clinical utility of these guidelines, given advances in microarray technology and prenatal screening, is largely unaddressed.
What does this study add?
This study demonstrates the diagnostic superiority of CMA by SNP microarray compared with karyotyping for prenatal diagnosis, regardless of the clinical indication for testing.
Objective
The American College of Obstetricians and Gynecologists (ACOG) and Society for Maternal‐Fetal Medicine (SMFM) recommend chromosomal microarray analysis (CMA) for prenatal diagnosis in cases with 1 or more fetal structural abnormalities. For patients who elect prenatal diagnosis and have a structurally normal fetus, either microarray or karyotype is recommended. This study evaluates the frequency of clinically significant chromosomal abnormalities (CSCA) that would have been missed if all patients offered the choice between CMA and karyotyping chose karyotyping.
Methods
A total of 3223 prenatal samples undergoing CMA were evaluated. Cases were categorized into 2 groups: those that met ACOG guidelines for CMA versus those that met ACOG guidelines for either CMA or karyotype.
Results
Of the 3223 cases, 1475 (45.8%) met ACOG recommendations for CMA, and 1748 (54.2%) met recommendations for either CMA or karyotype. In patients who could have elected either CMA or karyotype, 2.5% had CSCA that would have been missed if the patient had elected to pursue karyotype.
Conclusion
This study suggests that 2.5% of patients will have a CSCA that may be missed if the guidelines continue to suggest that CMA and karyotyping have equivalent diagnostic value for patients without a fetal structural abnormality.
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