Single-cell mass cytometry adapted to measurements of the cell cycle

Mass cytometry is a recently introduced technology that utilizes transition element isotope-tagged antibodies for protein detection on a single-cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently, a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The current study describes approaches to delineate cell cycle stages utilizing 5-iodo-2-deoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage.     

Sediments and sedimentary history of Lake Attersee (Salzkammergut,Austria)

Attersee represents a good example of a lake situated in the Northern forelands of the Northern Calcareous Alps and influenced by different sediment-supplying processes during the postglacial. Several compounds, of different origin, form the sediments of the basin. Clastics which are mainly composed of dolomites derive from the Northern Calcareous Alps. Clastic input of organic and inorganic particles is accomplished by rivers and landslides. They are responsible for the main input of siliciclasts like quartz, feldspar and mica. A high proportion of the sediment results from autochthonous biogenic carbonate precipitation. In the shallow sublittoral areas of the northern part of the lake benthic decalcification caused by encrusting macro- and micro-phytes is dominant, while in the southern and central parts of the lake epilimnetic decalcification caused by the blooming of phytoplancton is more important during summer. The total biogenic calcium carbonate production reaches about 11 000 to 12 000 metric tons a year.Nutrients and residues of cyanophytes (Oscillatoria rubescens) deriving from the eutrophic lake Mondsee were washed into lake Attersee by the Mondseeache. High amount of phosphorus in the sediments of the southern basin depicts local eutrophication in the mouth area of the Mondseeache. The average sedimentation rate in lake Attersee can be determined by different dating methods. Sedimentation rates increased during the last 110 years from 1 mm a year to 1.8–2 mm a year as a result of human activities. Five main phases in the postglacial sedimentary history can be recognized: Würm moraines and finely banded varves (before 13 000 B.P.), the early Attersee stage (from 13 000 B.P. up to 1200 B.P.), and the later Attersee stage after the Bavarian colonization (from 1200 B.P. on). Using heavy metal and isotope analyses the sedimentary history can be reconstructed in more detail for the last 100 years.     

Reference levels of the tumor markers carcinoembryonic antigen, the carbohydrate antigens 19-9 and 72-4, and cytokeratin fragment 19 using the Elecsys Relecsys 1010 analyzer in a normal population in Kuwait. The importance of the determination of local reference levels

The tumor markers CEA, CA 19-9, CA 72-4 and CYFRA 21-1 were analyzed in a group of apparently healthy subjects (n=232) in Kuwait using the Elecsys Relecsys 1010 analyzer. The distribution of the tumour marker levels was analyzed separately in Kuwaitis (n=103), non-Kuwaitis (n=129), smokers (n=68), non-smokers (n=164), males (n=138) and females (n=94). The distribution of CEA was significantly different in Kuwaitis vs. non-Kuwaitis in the total population (p=0.033) and in non-smokers (p=0.049); in males vs. females in the total population (p<0.0001) and in non-smokers (p=0.0002); and in smokers vs. non-smokers in the total population (p<0.0001) using the non-parametric Mann-Whitney U test. None of the other tumour markers showed significant differences in the subgroups. The upper reference level was defined as the 95th percentile of the normal values in each group. A higher reference level of CEA was observed in smokers (vs. non-smokers) in the total population. Also higher reference levels of CEA were observed in males (vs. females) both in the total population and in non-smokers. In the total population the respective reference levels were: CEA: 4.4 microg/L, CA 19-9: 35 kU/L, CA 72.4: 2.4 kU/L, and CYFRA 21.1: 2.1 microg/L. These results were compared with data in the kit inserts and literature data. The impact of 95th percentiles in a local heterogeneous population is discussed.     

Echinococcus multilocularis: inhibition of murine neutrophil and macrophage chemotaxis

Resident peritoneal neutrophils and macrophages from mice infected with 50 +/- 5 cysts of Echinococcus multilocularis were collected at 2, 4, 6, 8, 10, and 16 weeks postinfection. Their ability to respond and migrate to purified parasite larval antigens or endotoxin-activated mouse serum (EAMS) in comparison to normal peritoneal cells from uninfected mice was tested in vitro using Boyden chambers. Early in the infection, both cell types responded to the specific and nonspecific chemoattractants as the control group. However, at 8 and 10 weeks postinfection, the neutrophils and macrophages lost their response to parasite antigens but retained their ability to migrate to EAMS. Toward the 12th and 16th week postinfection, both cell types lost their ability to migrate to the specific as well as the nonspecific factors. The data presented suggest that the cellular mechanisms of recognition and chemotaxis in mice infected with alveolar hydatid cysts are impaired.     

Continuous Metabolic Syndrome Scores for Children Using Salivary Biomarkers

Background

Binary definitions of the metabolic syndrome based on the presence of a particular number of individual risk factors are limited, particularly in the pediatric population. To address this limitation, we aimed at constructing composite and continuous metabolic syndrome scores (cmetS) to represent an overall measure of metabolic syndrome (MetS) in a large cohort of metabolically at-risk children, focusing on the use of the usual clinical parameters (waist circumference (WC) and systolic blood pressure (SBP), supplemented with two salivary surrogate variables (glucose and high density lipoprotein cholesterol (HDLC). Two different approaches used to create the scores were evaluated in comparison.

Methods

Data from 8,112 Kuwaiti children (10.00 ± 0.67 years) were used to construct two cmetS for each subject. The first cmetS (cmetS-Z) was created by summing standardized residuals of each variable regressed on age and gender; and the second cmetS (cmetS-PCA) was defined as the first principal component from gender-specific principal component analysis based on the four variables.

Results

There was a graded relationship between both scores and the number of adverse risk factors. The areas under the curve using cmetS-Z and cmetS-PCA as predictors for severe metabolic syndrome (defined as the presence of ≥3 metabolic risk factors) were 0.935 and 0.912, respectively. cmetS-Z was positively associated with WC, SBP, and glucose, but inversely associated with HDLC. Except for the lack of association with glucose, cmetS-PCA was similar to cmetS-Z in boys, but had minimum loading on HDLC in girls. Analysis using quantile regression showed an inverse association of fitness level with cmetS-PCA (p = 0.001 for boys; p = 0.002 for girls), and comparison of cmetS-Z and cmetS-PCA suggested that WC and SBP were main contributory components. Significant alterations in the relationship between cmetS and salivary adipocytokines were demonstrated in overweight and obese children as compared to underweight and normal-weight children.

Conclusion

We have derived continuous summary scores for MetS from a large-scale pediatric study using two different approaches, incorporating salivary measures as surrogate for plasma measures. The derived scores were viable expressions of metabolic risk, and can be utilized to study the relationships of MetS with various aspects of the metabolic disease process.     

Induction, in vivo and in vitro, of macrophage membrane interleukin-1 by adjuvant-active synthetic muramyl peptides

Recent evidence has shown that a membrane form of interleukin-1 (IL-1) serves as a necessary signal for antigen presentation, leading to T-cell activation. The synthetic immunostimulant muramyl dipeptide (MDP) is known to induce secretion of IL-1 and its adjuvant effect was found to be mediated through enhancement of T-helper cells. We have investigated the ability of MDP and 19 other adjuvant-active or -inactive MDP analogs and derivatives to induce membrane IL-1 in mouse peritoneal macrophages. Enhancement in vitro of membrane expression and secretion of IL-1 in fresh or aged cultures of macrophages was observed after stimulation with MDP or with adjuvant-active but not with adjuvant-inactive muramyl peptides. Administration in vivo of adjuvant-active doses of MDP or of any of 12 other active analogs induced high levels of macrophage membrane IL-1 detected by the lymphocyte-activating factor assay. This effect was not observed when 7 other adjuvant-inactive derivatives were used. Moreover, under conditions where MDP did not exert an adjuvant effect, this immunomodulator was found to be incapable of inducing the expression of macrophage membrane IL-1. These results demonstrate a very high correlation between the ability to induce membrane IL-1 and the adjuvant activity of muramyl peptides. The correlation was observed irrespective of other biological effects of the synthetic adjuvants such as pyrogenicity and/or anti-infectious activity.     

Sequence and analysis of a whole genome from Kuwaiti population subgroup of Persian ancestry

Background

The 1000 Genome project paved the way for sequencing diverse human populations. New genome projects are being established to sequence underrepresented populations helping in understanding human genetic diversity. The Kuwait Genome Project an initiative to sequence individual genomes from the three subgroups of Kuwaiti population namely, Saudi Arabian tribe; “tent-dwelling” Bedouin; and Persian, attributing their ancestry to different regions in Arabian Peninsula and to modern-day Iran (West Asia). These subgroups were in line with settlement history and are confirmed by genetic studies. In this work, we report whole genome sequence of a Kuwaiti native from Persian subgroup at >37X coverage.

Results

We document 3,573,824 SNPs, 404,090 insertions/deletions, and 11,138 structural variations. Out of the reported SNPs and indels, 85,939 are novel. We identify 295 ‘loss-of-function’ and 2,314 ’deleterious’ coding variants, some of which carry homozygous genotypes in the sequenced genome; the associated phenotypes include pharmacogenomic traits such as greater triglyceride lowering ability with fenofibrate treatment, and requirement of high warfarin dosage to elicit anticoagulation response. 6,328 non-coding SNPs associate with 811 phenotype traits: in congruence with medical history of the participant for Type 2 diabetes and β-Thalassemia, and of participant’s family for migraine, 72 (of 159 known) Type 2 diabetes, 3 (of 4) β-Thalassemia, and 76 (of 169) migraine variants are seen in the genome. Intergenome comparisons based on shared disease-causing variants, positions the sequenced genome between Asian and European genomes in congruence with geographical location of the region. On comparison, bead arrays perform better than sequencing platforms in correctly calling genotypes in low-coverage sequenced genome regions however in the event of novel SNP or indel near genotype calling position can lead to false calls using bead arrays.

Conclusions

We report, for the first time, reference genome resource for the population of Persian ancestry. The resource provides a starting point for designing large-scale genetic studies in Peninsula including Kuwait, and Persian population. Such efforts on populations under-represented in global genome variation surveys help augment current knowledge on human genome diversity.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1233-x) contains supplementary material, which is available to authorized users.