Proteomic analysis of necroptotic extracellular vesicles

Necroptosis is a regulated and inflammatory form of cell death. We, and others, have previously reported that necroptotic cells release extracellular vesicles (EVs). We have found that necroptotic EVs are loaded with proteins, including the phosphorylated form of the key necroptosis-executing factor, mixed lineage kinase domain-like kinase (MLKL). However, neither the exact protein composition, nor the impact, of necroptotic EVs have been delineated. To characterize their content, EVs from necroptotic and untreated U937 cells were isolated and analyzed by mass spectrometry-based proteomics. A total of 3337 proteins were identified, sharing a high degree of similarity with exosome proteome databases, and clearly distinguishing necroptotic and control EVs. A total of 352 proteins were significantly upregulated in the necroptotic EVs. Among these were MLKL and caspase-8, as validated by immunoblot. Components of the ESCRTIII machinery and inflammatory signaling were also upregulated in the necroptotic EVs, as well as currently unreported components of vesicle formation and transport, and necroptotic signaling pathways. Moreover, we found that necroptotic EVs can be phagocytosed by macrophages to modulate cytokine and chemokine secretion. Finally, we uncovered that necroptotic EVs contain tumor neoantigens, and are enriched with components of antigen processing and presentation. In summary, our study reveals a new layer of regulation during the early stage of necroptosis, mediated by the secretion of specific EVs that influences the microenvironment and may instigate innate and adaptive immune responses. This study sheds light on new potential players in necroptotic signaling and its related EVs, and uncovers the functional tasks accomplished by the cargo of these necroptotic EVs.Subject terms: Necroptosis, Cell death and immune response     

Rational confederation of genes and diseases: NGS interpretation via GeneCards,MalaCards and VarElect

Background

A key challenge in the realm of human disease research is next generation sequencing (NGS) interpretation, whereby identified filtered variant-harboring genes are associated with a patient’s disease phenotypes. This necessitates bioinformatics tools linked to comprehensive knowledgebases. The GeneCards suite databases, which include GeneCards (human genes), MalaCards (human diseases) and PathCards (human pathways) together with additional tools, are presented with the focus on MalaCards utility for NGS interpretation as well as for large scale bioinformatic analyses.

Results

VarElect, our NGS interpretation tool, leverages the broad information in the GeneCards suite databases. MalaCards algorithms unify disease-related terms and annotations from 69 sources. Further, MalaCards defines hierarchical relatedness—aliases, disease families, a related diseases network, categories and ontological classifications. GeneCards and MalaCards delineate and share a multi-tiered, scored gene-disease network, with stringency levels, including the definition of elite status—high quality gene-disease pairs, coming from manually curated trustworthy sources, that includes 4500 genes for 8000 diseases. This unique resource is key to NGS interpretation by VarElect. VarElect, a comprehensive search tool that helps infer both direct and indirect links between genes and user-supplied disease/phenotype terms, is robustly strengthened by the information found in MalaCards. The indirect mode benefits from GeneCards’ diverse gene-to-gene relationships, including SuperPaths—integrated biological pathways from 12 information sources. We are currently adding an important information layer in the form of “disease SuperPaths”, generated from the gene-disease matrix by an algorithm similar to that previously employed for biological pathway unification. This allows the discovery of novel gene-disease and disease–disease relationships. The advent of whole genome sequencing necessitates capacities to go beyond protein coding genes. GeneCards is highly useful in this respect, as it also addresses 101,976 non-protein-coding RNA genes. In a more recent development, we are currently adding an inclusive map of regulatory elements and their inferred target genes, generated by integration from 4 resources.

Conclusions

MalaCards provides a rich big-data scaffold for in silico biomedical discovery within the gene-disease universe. VarElect, which depends significantly on both GeneCards and MalaCards power, is a potent tool for supporting the interpretation of wet-lab experiments, notably NGS analyses of disease. The GeneCards suite has thus transcended its 2-decade role in biomedical research, maturing into a key player in clinical investigation.

     

Standing on the shoulders of giants: young aphids piggyback on adults when searching for a host plant

Background

Upon the detection of imminent peril, pea aphids (Acyrthosiphon pisum) often drop off their host plant. Dropping in response to insect enemies is intermittent in nature, but when a mammalian herbivore feeds on their host plant, a large mixed-age group of aphids usually drops off the plant at once. Aphids that reach the ground are confronted with new, hostile environmental conditions and must therefore quickly walk toward a suitable host plant. The longer it takes an aphid to reach a host plant, the more it is exposed to the risks of starvation, desiccation and predation.

Results

We found that young nymphs, which have limited mobility and high mortality on the ground, quickly climb on conspecific (not necessarily parental) adults and cling to them before the latter start walking in search of a plant. This “riding” behavior is likely to be adaptive for the nymphs, for it shortens their journey and the time they spend off a host plant. Adults however, seem to be irritated by the riding nymphs, as they often actively try to remove them.

Conclusions

After dropping from the host plant, young aphid nymphs travel at least part of the way back to a plant on the backs of adults. For the riding behavior to take place, nymphs need to successfully find adults and withstand removal attempts.

     

Physiological evidence for central modulation of voice tremor

The present report presents an attempt to define the physiological parameter used to describe “voice tremor” in psychological stress evaluating machines, and to find its sources. This parameter was found to be a low frequency (5–20 Hz) random process which frequency modulates the vocal cord waveform and (independently) affects the frequency range of the third speech formant. The frequency variations in unstressed speakers were found to be the result of forced muscular undulations driven by central nervous signals and not of a passive resonant phenomenon. In this paper various physiological and clinical experiments which lead to the above conclusions are discussed. a) It is shown that induced muscular activity in the vocal tract and vocal cord regions can generate tremor in the voice. b) It is shown that relaxed subjects exhibit significant tremor correlation between spontaneously generated speech and EMG, with the EMG leading the speech tremor. c) Tremor in the electrical activity recorded from muscles overlapping vocal tract area was correlated with third formant demodulated signal and vocal cord demodulated pitch tremor was correlated with first formant demodulated tremor. d) Enhanced tremor was found in Parkinson patients and diminished tremor in patients with some traumatic brain injuries.     

The effects of intermittent warm-up on 7-9 year-old boys.

Twelve boys, 7-9 years old, who, by use of questionnaires, were found to be ignorant of the concept of "warm-up", performed maximal aerobic and anaerobic tasks 4 min after completing a 15 min intermittent warm-up (WU) (30 sec treadmill run, 30 sec pause). The WU required some 60% of the individual's VO2max, and raised rectal temp. by 0.52 +/- 0.19 degrees C. The aerobic criterion task (CT) was a one-stage bicycle ride at a load predetermined to exhaust the subject after 4 min. The anaerobic CT was a 30 sec all-out ride against resistance of 35 gm/kg BW. Subjects also performed both CT's without any WU. VO2max (aerobic CT), HRmax, as well as total mechanical work output achieved during the aerobic CT, were significantly higher with WU, compared with the non-WU sessions. During the anaerobic CT total revolutions, total power output, as well as peak HR, were significantly higher following WU. It is suggested that the benefits of intermittent WU, as used in this study in young children, can be attributed to physiological rather than psychological mechanisms.     

Location of amino acid and carbohydrate transport sites in the surface membrane of normal and transformed mammalian cells

Summary Amino acid and carbohydrate transport in normal and malignant transformed hamster cells was studied after binding of the protein Concanavalin A (Con. A) to the surface membrane. Experimental conditions were used so that a similar number of Con. A molecules were bound to both types of cells. The transport of amino acids was inhibited after Con. A binding in the transformed cells but not in normal cells. This was found with the metabolizable amino acidsl-leucine,l-arginine,l-glutamic acid, andl-glutamine, and with the non-metabolizable amino acids cycloleucine and -aminoisobutyric acid. Transport ofd-glucose andd-galactose was more inhibited by Con. A in transformed than in normal cells, and in both types of cellsd-glucose was inhibited more thand-galactose. The inhibition by Con. A on transport was specific, since there was no effect on the transport ofl-fucose in either normal or transformed cells. Con. A also did not effect the entry of 3-0-methyl-d-glucose.These observations can be used to locate amino acid and carbohydrate transport sites in the surface membrane in relation to the binding sites for Con. A. The results indicate that Con. A sites are associated in normal cells with transport sites ford-glucose and to a lesser extentd-galactose, and in transformed cells with transport sites for amino acids and to a greater extent than in normal cells withd-glucose andd-galactose. Malignant transformation of normal cells therefore results in a change in the location of amino acid and carbohydrate transport sites in the surface membrane in relation to the binding sites for Con. A.     

Effect of an iron supplement on body iron status and aerobic capacity of young training women

Serum iron deficiency has a high incidence in female athletes. We investigated the effects of a daily oral iron supplement, (160 mg) administered during an intensive 7-week physical training programme, on body iron status, and the maximal aerobic capacity (VO2max) of 13 women (group A) compared to 15 who took a placebo (group B). The subjects were 19 years old. Blood samples were obtained before training began and on days 1, 7, 21 and 42 of training. They were analysed for packed cell volume (PVC) and for haemoglobin (Hb), 2,3-diphosphoglycerate (2,3-DPG), haptoglobin, iron and ferritin concentrations. The VO2max was measured on days 0, 21 and 42 of training. Following 21 days of training Hb, PCV and ferritin were significantly higher (P less than or equal to 0.01) in group A compared to group B. Over the training period Hb rose by 9.3% and 2.4% in groups A and B, respectively. At the end of training 66% of group B exhibited ferritin concentrations below 10 ng.ml-1, while none of group A had such low values. Mean VO2max of group A had increased by 7.5% following 21 days of training (P less than or equal to 0.01) and by 15.3% after 42 days. No appreciable increase in VO2max had occurred in group B by day 21 (significantly lower than VO2max of group A; P less than or equal to 0.05), however by day 42 it had increased by 14.3% (P less than or equal to 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

13C nuclear magnetic resonance and gas chromatography-mass spectrometry studies of carbon metabolism in the actinomycin D producer Streptomyces parvulus by use of 13C-labeled precursors.

Fructose and glutamate metabolism was monitored in cell suspensions of streptomyces parvulus by 13C nuclear magnetic resonance. The experiments were performed for cells grown with various 13C sources in a growth medium containing D-[U-13C]fructose, L-[13C]glutamate, or L-[U-13C]aspartate and with nonlabeled precursors to compare intracellular pools in S. parvulus cells at different periods of the cell life cycle. The transport of fructose into the cells was biphasic in nature; during rapid transport, mannitol, fructose, and glucose 6-phosphate were accumulated intracellularly, whereas during the passive diffusion of fructose, the intracellular carbohydrate pool comprised mainly trehalose (1,1'-alpha-alpha-D-glucose). The regulation of fructokinase activity by the intracellular intermediates may play an important role in fructose catabolism in S. parvulus. Transaldolase activity in S. parvulus was determined from the 13C nuclear magnetic resonance labeling pattern of trehalose carbons obtained from cells grown in medium containing either L-[U-13C]aspartate or L-[U-13C]glutamate. Only carbons 4, 5, and 6 of the disaccharide were labeled. Isotopomer analysis of the trehalose carbons led us to conclude that the flux through the reverse glycolytic pathway, condensation of glyceraldehyde 3-phosphate with dihydroxyacetone phosphate, makes at best a minor contribution to the 13C-labeled glucose units observed in trehalose. The pentose pathway and transaldolase activity can explain the labeling pattern of 4,5,6-13C3 of trehalose. Moreover, the transfer of the 13C label of L-[U-13C]aspartate into the different isotopomers of trehalose C4, C5, and C6 by the transaldolase activity allowed us to calculate the relative fluxes from oxaloacetate via gluconeogenesis and through the tricarboxylic acid cycle. The ratio of the two fluxes is approximately 1. However, the main carbon source for trehalose synthesis in S. parvulus is fructose and not glutamate or aspartate. The 13C enrichment and isotopomer population, measured by nuclear magnetic resonance and gas chromatography-mass spectrometry, of the actinomycin D peptide ring enabled us to specify the origins of the five amino acids of actinomycin D. Threonine and proline exhibited isotopomer populations similar to that of the extracellular L-[13C]glutamate, indicating that protein catabolism is the origin of their 13C label, whereas the isotopomer populations of sarcosine and N-methylvaline were similar to those of the new intracellular pool of S. parvulus that originated from D-[U-13C]fructose during the production of actinomycin D.