Gemin8 is a novel component of the survival motor neuron complex and functions in small nuclear ribonucleoprotein assembly

The survival motor neuron (SMN) protein is the product of the spinal muscular atrophy disease gene. SMN and Gemin2-7 proteins form a large macromolecular complex that localizes in the cytoplasm as well as in the nucleoplasm and in nuclear Gems. The SMN complex interacts with several additional proteins and likely functions in multiple cellular pathways. In the cytoplasm, a subset of SMN complexes containing unrip and Sm proteins mediates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Here, by mass spectrometry analysis of SMN complexes purified from HeLa cells, we identified a novel protein that is evolutionarily conserved in metazoans, and we named it Gemin8. Co-immunoprecipitation and immunolocalization experiments demonstrated that Gemin8 is associated with the SMN complex and is localized in the cytoplasm and in the nucleus, where it is highly concentrated in Gems. Gemin8 interacts directly with the Gemin6-Gemin7 heterodimer and, together with unrip, these proteins form a heteromeric subunit of the SMN complex. Gemin8 is also associated with Sm proteins, and Gemin8-containing SMN complexes are competent to carry out snRNP assembly. Importantly, RNA interference experiments indicate that Gemin8 knock-down impairs snRNP assembly, and Gemin8 expression is down-regulated in cells with low levels of SMN. These results demonstrate that Gemin8 is a novel integral component of the SMN complex and extend the repertoire of cellular proteins involved in the pathway of snRNP biogenesis.     

The inhibitors of cyclin-dependent kinases and GSK-3β enhance osteoclastogenesis

Osteoclasts are multinucleated cells with bone resorption activity that is crucial for bone remodeling. RANK‐RANKL (receptor activator of nuclear factor κB ligand) signaling has been shown as a main signal pathway for osteoclast differentiation. However, the molecular mechanism and the factors regulating osteoclastogenesis remain to be fully understood. In this study, we performed a chemical genetic screen, and identified a Cdks/GSK-3β (cyclin-dependent kinases/glycogen synthase kinase 3β) inhibitor, kenpaullone, and two Cdks inhibitors, olomoucine and roscovitine, all of which significantly enhance osteoclastogenesis of RAW264.7 cells by upregulating NFATc1 (nuclear factor of activated T cells, cytoplasmic 1) levels. We also determined that the all three compounds increase the number of osteoclast differentiated from murine bone marrow cells. Furthermore, the three inhibitors, especially kenpaullone, promoted maturation of cathepsin K, suggesting that the resorption activity of the resultant osteoclasts is also activated. Our findings indicate that inhibition of GSK-3β and/or Cdks enhance osteoclastogenesis by modulating the RANK–RANKL signaling pathway.     

Higher plant photosystem II light-harvesting antenna, not the reaction center, determines the excited-state lifetime-both the maximum and the nonphotochemically quenched

The maximum chlorophyll fluorescence lifetime in isolated photosystem II (PSII) light-harvesting complex (LHCII) antenna is 4 ns; however, it is quenched to 2 ns in intact thylakoid membranes when PSII reaction centers (RCIIs) are closed (Fm). It has been proposed that the closed state of RCIIs is responsible for the quenching. We investigated this proposal using a new, to our knowledge, model system in which the concentration of RCIIs was highly reduced within the thylakoid membrane. The system was developed in Arabidopsis thaliana plants under long-term treatment with lincomycin, a chloroplast protein synthesis inhibitor. The treatment led to 1), a decreased concentration of RCIIs to 10% of the control level and, interestingly, an increased antenna component; 2), an average reduction in the yield of photochemistry to 0.2; and 3), an increased nonphotochemical chlorophyll fluorescence quenching (NPQ). Despite these changes, the average fluorescence lifetimes measured in Fm and Fm' (with NPQ) states were nearly identical to those obtained from the control. A 77 K fluorescence spectrum analysis of treated PSII membranes showed the typical features of preaggregation of LHCII, indicating that the state of LHCII antenna in the dark-adapted photosynthetic membrane is sufficient to determine the 2 ns Fm lifetime. Therefore, we conclude that the closed RCs do not cause quenching of excitation in the PSII antenna, and play no role in the formation of NPQ.     

The 1-deoxy-d-xylulose 5-phosphate synthase gene co-localizes with a major QTL affecting monoterpene content in grapevine

Muscat flavor is a relevant trait both in winemaking and in fresh grape consumption. From a chemical point of view, it is strongly related to the accumulation of monoterpenes in berries. However, knowledge of the genetic mechanisms underlying its regulation is still limited. The objective of this study was to dissect the genetic determinism of aroma in grapevine by applying the analysis of quantitative trait loci (QTL) and the candidate gene (CG) approach. Two F₁ segregating progenies were evaluated through high-resolution gas chromatography–mass spectrometry (HRGC–MS) for the amounts of individual monoterpenes over 3 and 2 years. In the Italia × Big Perlon cross 34 CGs, chosen according to gene ontology (GO) terms, were placed on a complete map and tested for linkage with QTLs for linalool, nerol and geraniol levels. Two CGs mapped within a QTL for linalool content on LG 10. A third one co-localized with a major QTL for the level of the three monoterpenes on LG 5; this gene encodes 1-deoxy-d-xylulose 5-phosphate synthase (DXS), which is the first enzyme in the plastidial pathway of terpene biosynthesis. Depending on these findings, we report the first in silico analysis of grapevine DXS genes based on the whole genome sequence. Further research on the functional significance of these associations might help to understand the genetic control of Muscat flavor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Battilana and L. Costantini equally contributed to the work.     

Ethiopian mitochondrial DNA heritage: tracking gene flow across and around the gate of tears

Approximately 10 miles separate the Horn of Africa from the Arabian Peninsula at Bab-el-Mandeb (the Gate of Tears). Both historic and archaeological evidence indicate tight cultural connections, over millennia, between these two regions. High-resolution phylogenetic analysis of 270 Ethiopian and 115 Yemeni mitochondrial DNAs was performed in a worldwide context, to explore gene flow across the Red and Arabian Seas. Nine distinct subclades, including three newly defined ones, were found to characterize entirely the variation of Ethiopian and Yemeni L3 lineages. Both Ethiopians and Yemenis contain an almost-equal proportion of Eurasian-specific M and N and African-specific lineages and therefore cluster together in a multidimensional scaling plot between Near Eastern and sub-Saharan African populations. Phylogeographic identification of potential founder haplotypes revealed that approximately one-half of haplogroup L0–L5 lineages in Yemenis have close or matching counterparts in southeastern Africans, compared with a minor share in Ethiopians. Newly defined clade L6, the most frequent haplogroup in Yemenis, showed no close matches among 3,000 African samples. These results highlight the complexity of Ethiopian and Yemeni genetic heritage and are consistent with the introduction of maternal lineages into the South Arabian gene pool from different source populations of East Africa. A high proportion of Ethiopian lineages, significantly more abundant in the northeast of that country, trace their western Eurasian origin in haplogroup N through assorted gene flow at different times and involving different source populations.     

Optimization of SPECT-CT Hybrid Imaging Using Iterative Image Reconstruction for Low-Dose CT: A Phantom Study

Background

Hybrid imaging combines nuclear medicine imaging such as single photon emission computed tomography (SPECT) or positron emission tomography (PET) with computed tomography (CT). Through this hybrid design, scanned patients accumulate radiation exposure from both applications. Imaging modalities have been the subject of long-term optimization efforts, focusing on diagnostic applications. It was the aim of this study to investigate the influence of an iterative CT image reconstruction algorithm (ASIR) on the image quality of the low-dose CT images.

Methodology/Principal Findings

Examinations were performed with a SPECT-CT scanner with standardized CT and SPECT-phantom geometries and CT protocols with systematically reduced X-ray tube currents. Analyses included image quality with respect to photon flux. Results were compared to the standard FBP reconstructed images. The general impact of the CT-based attenuation maps used during SPECT reconstruction was examined for two SPECT phantoms. Using ASIR for image reconstructions, image noise was reduced compared to FBP reconstructions for the same X-ray tube current. The Hounsfield unit (HU) values reconstructed by ASIR were correlated to the FBP HU values(R² ≥ 0.88) and the contrast-to-noise ratio (CNR) was improved by ASIR. However, for a phantom with increased attenuation, the HU values shifted for low X-ray tube currents I ≤ 60 mA (p ≤ 0.04). In addition, the shift of the HU values was observed within the attenuation corrected SPECT images for very low X-ray tube currents (I ≤ 20 mA, p ≤ 0.001).

Conclusion/Significance

In general, the decrease in X-ray tube current up to 30 mA in combination with ASIR led to a reduction of CT-related radiation exposure without a significant decrease in image quality.     

Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation

Smad7 is an inhibitory Smad that acts as a negative regulator of signaling by the transforming growth factor-beta (TGF-beta) superfamily proteins. Smad7 is induced by TGF-beta, stably interacts with activated TGF-beta type I receptor (TbetaR-I), and interferes with the phosphorylation of receptor-regulated Smads. Here we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic protein-specific Smads, also interacts with Smad7 and induces Smad7 ubiquitination and translocation into the cytoplasm. In addition, Smurf1 associates with TbetaR-I via Smad7, with subsequent enhancement of turnover of TbetaR-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of TbetaR-I through recruitment of an E3 ligase to the receptor.     

Near-infrared optical imaging of integrin alphavbeta3 in human tumor xenografts

In vivo optical imaging is potentially useful for evaluating the presence of tumor markers that are targets of molecular medicine. Here we report the synthesis and characterization of integrin alphavbeta3-targeted peptide cyclo(Lys-Arg-Gly-Asp-Phe) [c(KRGDf )] labeled with fluorescence dyes with wavelength spanning from the visible/near infrared (Cy5.5) to the true near infrared (IRDye800) for optical imaging. In vitro, the peptide-dye conjugates bound specifically to tumor cells expressing alphavbeta3. When administered intravenously into mice at a dose of 6 nmol /mouse, the conjugates accumulated in tumors expressing alphavbeta3. The tumor-to-background ratios for human KS1767 Kaposi's sarcoma in mice injected with Cy5.5-c(KRGDf ) and Cy5.5 were 5.5 and 1.5, respectively. Preinjection of c(KRGDf ) blocked the uptake of Cy5.5-c(KRGDf ) in tumors by 89%. In alphavbeta3-positive M21 and alphavbeta3-negative M21-L human melanoma, fluorescence intensity in the tumor of mice injected with IRDye800 - c(KRGDf ) was 2.3 and 1.3 times that in normal tissue, respectively. Dynamic imaging revealed that Cy5.5- c(KRGDf ) was rapidly taken up by KS1767 tumor immediately after bolus injection. The rate of its uptake in the tumor was reduced by preinjection of c(KRGDf ) in an interval time-dependent manner. Our data suggest that near-infrared fluorescence imaging may be applied to the detection of tumors expressing integrin alphavbeta3 and to the assessment of the optimal biological dose and schedule of targeted therapies.     

Is mass spectrometry ready for proteome-wide protein expression analysis?

Recent advances in mass spectrometry will soon allow routine analysis of protein expression levels. How close are we to true quantitative proteomics?