Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3nm precision using aberration-corrected scanning transmission electron microscopy

Three-dimensional (3D) maps of proteins within the context of whole cells are important for investigating cellular function. However, 3D reconstructions of whole cells are challenging to obtain using conventional transmission electron microscopy (TEM). We describe a methodology to determine the 3D locations of proteins labeled with gold nanoparticles on whole eukaryotic cells. The epidermal growth factor receptors on COS7 cells were labeled with gold nanoparticles, and critical-point dried whole-mount cell samples were prepared. 3D focal series were obtained with aberration-corrected scanning transmission electron microscopy (STEM), without tilting the specimen. The axial resolution was improved with deconvolution. The vertical locations of the nanoparticles in a whole-mount cell were determined with a precision of 3nm. From the analysis of the variation of the axial positions of the labels we concluded that the cellular surface was ruffled. To achieve sufficient stability of the sample under electron beam irradiation during the recording of the focal series, the sample was carbon coated. A quantitative method was developed to analyze the stability of the ultrastructure after electron beam irradiation using TEM. The results of this study demonstrate the feasibility of using aberration-corrected STEM to study the 3D nanoparticle distribution in whole cells.     

An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells

An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti-stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.     

Extent of height variability explained by known height-associated genetic variants in an isolated population of the Adriatic coast of Croatia

Background

Human height is a classical example of a polygenic quantitative trait. Recent large-scale genome-wide association studies (GWAS) have identified more than 200 height-associated loci, though these variants explain only 2∼10% of overall variability of normal height. The objective of this study was to investigate the variance explained by these loci in a relatively isolated population of European descent with limited admixture and homogeneous genetic background from the Adriatic coast of Croatia.

Methodology/Principal Findings

In a sample of 1304 individuals from the island population of Hvar, Croatia, we performed genome-wide SNP typing and assessed the variance explained by genetic scores constructed from different panels of height-associated SNPs extracted from five published studies. The combined information of the 180 SNPs reported by Lango Allen el al. explained 7.94% of phenotypic variation in our sample. Genetic scores based on 20∼50 SNPs reported by the remaining individual GWA studies explained 3∼5% of height variance. These percentages of variance explained were within ranges comparable to the original studies and heterogeneity tests did not detect significant differences in effect size estimates between our study and the original reports, if the estimates were obtained from populations of European descent.

Conclusions/Significance

We have evaluated the portability of height-associated loci and the overall fitting of estimated effect sizes reported in large cohorts to an isolated population. We found proportions of explained height variability were comparable to multiple reference GWAS in cohorts of European descent. These results indicate similar genetic architecture and comparable effect sizes of height loci among populations of European descent.     

Predictive and prognostic impact of TP53 mutations and MDM2 promoter genotype in primary breast cancer patients treated with epirubicin or paclitaxel

Background

TP53 mutations have been associated with resistance to anthracyclines but not to taxanes in breast cancer patients. The MDM2 promoter single nucleotide polymorphism (SNP) T309G increases MDM2 activity and may reduce wild-type p53 protein activity. Here, we explored the predictive and prognostic value of TP53 and CHEK2 mutation status together with MDM2 SNP309 genotype in stage III breast cancer patients receiving paclitaxel or epirubicin monotherapy.

Experimental Design

Each patient was randomly assigned to treatment with epirubicin 90 mg/m² (n = 109) or paclitaxel 200 mg/m² (n = 114) every 3rd week as monotherapy for 4–6 cycles. Patients obtaining a suboptimal response on first-line treatment requiring further chemotherapy received the opposite regimen. Time from last patient inclusion to follow-up censoring was 69 months. Each patient had snap-frozen tumor tissue specimens collected prior to commencing chemotherapy.

Principal Findings

While TP53 and CHEK2 mutations predicted resistance to epirubicin, MDM2 status did not. Neither TP53/CHEK2 mutations nor MDM2 status was associated with paclitaxel response. Remarkably, TP53 mutations (p = 0.007) but also MDM2 309TG/GG genotype status (p = 0.012) were associated with a poor disease-specific survival among patients having paclitaxel but not patients having epirubicin first-line. The effect of MDM2 status was observed among individuals harbouring wild-type TP53 (p = 0.039) but not among individuals with TP53 mutated tumors (p>0.5).

Conclusion

TP53 and CHEK2 mutations were associated with lack of response to epirubicin monotherapy. In contrast, TP53 mutations and MDM2 309G allele status conferred poor disease-specific survival among patients treated with primary paclitaxel but not epirubicin monotherapy.     

Ion-swimming speed variation ofVibrio cholerae cells

In the present work we report the variation in swimming speed ofVibrio cholerae with respect to the change in concentration of sodium ions in the medium. We have also studied the variation in swimming speed with respect to temperature. We find that the swimming speed initially shows a linear increase with the increase of the sodium ions in the medium and then plateaus. The range within which the swimming speed attains saturation is approximately the same at different temperatures.     

Direct plant regeneration of curry leaf tree (<Emphasis Type="Italic">Murraya koenigii</Emphasis> koenig.), an aromatic plant

Summary An efficient protocol for plant regeneration from stem segments of Murraya koenigii was developed by culturing on Murashige and Skoog (MS) medium supplemented with 2.5 mg l⁻¹ benzyladenine (BA), 25 mgl⁻¹ adenine sulfate, 0.25 mgl⁻¹ indole-3-acetic acid (IAA), and 3% sucrose. The frequency of shoot bud regeneration was higher on similar medium in subsequent subcultures. The regenerated shoots were rooted on half-strength basal MS medium supplemented with 0.25–0.5 mgl⁻¹ IAA or 1-naphthaleneacetic acid (NAA) within 8–12 d of culture. The maximum percentage of rooting was obtained on MS medium supplemented with IAA and NAA, each at 0.25 mgl⁻¹. During acclimatization, 95% of rooted plantlets survived were grown normally under greenhouse conditions.