Aurora A kinase and its substrate TACC3 are required for central spindle assembly

Cell division entails a marked reorganization of the microtubule network to form the spindle, a molecular machine that ensures accurate chromosome segregation to the daughter cells. Spindle organization is highly dynamic throughout mitosis and requires the activity of several kinases and complex regulatory mechanisms. Aurora A (AurA) kinase is essential for the assembly of the metaphase bipolar spindle and, thus, it has been difficult to address its function during the last phases of mitosis. Here, we examine the consequences of inhibiting AurA in cells undergoing anaphase, and show that AurA kinase activity is necessary for the assembly of a robust central spindle during anaphase. We also identify TACC3 as an AurA substrate essential in central spindle formation.     

Does Vitamin D affects changes in volumetric bone mineral density and architecture in postmenopausal women after conservatively treated distal radius fractures?

Objective:We examined the role of vitamin D on volumetric bone mineral density (vBMD) and architecture during the first week’s post-fracture in postmenopausal women (PMW) with distal radial fractures (DRF) treated conservatively using peripheral Quantitative Computed Tomography (pQCT).Methods:Patients were classified into 2 groups according to initial median 25(OH)D level; Group A (25(OH)D ≥15 ng/ml) and group B (25(OH)D <15 ng/ml). All patients were followed for 12 weeks at three visits: baseline, 6 weeks and 12 weeks post fracture. pQCT was performed at baseline in fractured and contralateral non-fractured radius and at 6^th and 12^th week on the fractured side.Results:39 patients completed the protocol. Mean 25(OH)D levels were 15.60±7.35 ng/ml (3.5-41.7). Trabecular (trab) bone mineral content (BMC) and trabvBMD increased at 6 wk. vs. baseline (p<0.001). Cortical BMC, cortvBMD and cross- sectional area (CSA) progressively decreased (p<0.001) during the 12 weeks. There was no interaction between baseline 25(OH)D levels and changes in trabecular and cortical BMC, vBMD and CSA. Advanced age and higher CTX and P1NP were associated with higher cortical bone loss.Conclusion:Vitamin D deficiency does not affect the early architectural changes after a DRF. Advanced age and higher bone remodeling were associated with higher cortical bone loss, probably related to immobilization and independent of vitamin D levels.     

Role of Endothelial Progenitor Cells and Inflammatory Cytokines in Healing of Diabetic Foot Ulcers

Background

To evaluate changes in endothelial progenitor cells (EPCs) and cytokines in patients with diabetic foot ulceration (DFU) in association with wound healing.

Methods

We studied healthy subjects, diabetic patients not at risk of DFU, at risk of DFU and with active DFU. We prospectively followed the DFU patients over a 12-week period. We also investigated similar changes in diabetic rabbit and mouse models of wound healing.

Results

All EPC phenotypes except the kinase insert domain receptor (KDR)⁺CD133⁺ were reduced in the at risk and the DFU groups compared to the controls. There were no major EPC differences between the control and not at risk group, and between the at risk and DFU groups. Serum stromal-cell derived factor-1 (SDF-1) and stem cell factor (SCF) were increased in DFU patients. DFU patients who healed their ulcers had lower CD34⁺KDR⁺ count at visits 3 and 4, serum c-reactive protein (CRP) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at visit 1, interleukin-1 (IL-1) at visits 1 and 4. EPCs tended to be higher in both diabetic animal models when compared to their non-diabetic counterparts both before and ten days after wounding.

Conclusions

Uncomplicated diabetes does not affect EPCs. EPCs are reduced in patients at risk or with DFU while complete wound healing is associated with CD34⁺KDR⁺ reduction, suggesting possible increased homing. Low baseline CRP, IL-1α and GM-CSF serum levels were associated with complete wound healing and may potentially serve as prognostic markers of DFU healing. No animal model alone is representative of the human condition, indicating the need for multiple experimental models.     

Significantly increased CCL5/RANTES and CCR7 mRNA levels in localized scleroderma

Plaque-type morphea, the most common subtype of localized scleroderma (LS), is a connective tissue disease which is characterized by immunological dysregulation, vascular alterations, and skin fibrosis. We aimed to investigate the expression profiles of different cytokines and chemokines in patients with LS and healthy controls. Twenty patients with plaque-type morphea and 18 healthy controls were investigated. Skin biopsies were performed for real-time RT-PCR studies. Median mRNA of interleukin (IL)-6 was significantly higher expressed in LS than in normal skin. By contrast, median IL-1α mRNA levels were significantly decreased in LS as compared to controls. Median mRNA expression of CCL13, IL-1β, IL-2, and tumor necrosis factor-α did not significantly differ between LS lesions and healthy skin. However, we observed significantly increased median chemokine ligand 5/RANTES (CCL5/RANTES) and median chemokine receptor 7 (CCR7) mRNA expression in LS lesions as compared to healthy controls. CCL5/RANTES and CCR7 mRNA expression significantly correlated in LS lesions. We could confirm data of previous studies indicating that gene expression of IL-6 is up-regulated in LS lesions as compared to healthy skin. Moreover, we have shown for the first time a significant increase of mRNA levels of CCR7 and CCL5/RANTES in LS lesions indicating an important pathogenetic role of chemokines in LS.     

The study of the E-class SEPALLATA3-like MADS-box genes in wild-type and mutant flowers of cultivated saffron crocus (Crocus sativus L.) and its putative progenitors

To further understand flowering and flower organ formation in the monocot crop saffron crocus (Crocus sativus L.), we cloned four MIKC^c type II MADS-box cDNA sequences of the E-class SEPALLATA3 (SEP3) subfamily designated CsatSEP3a/b/c/c_as as well as the three respective genomic sequences. Sequence analysis showed that cDNA sequences of CsatSEP3 c and c_as are the products of alternative splicing of the CsatSEP3c gene. Bioinformatics analysis with putative orthologous sequences from various plant species suggested that all four cDNA sequences encode for SEP3-like proteins with characteristic motifs and amino acids, and highlighted intriguing sequence features. Phylogenetically, the isolated sequences were closest to the SEP3-like genes from monocots such as Asparagus virgatus, Oryza sativa, Zea mays, and the dicot Arabidopsis SEP3 gene. All four isolated C. sativus sequences were strongly expressed in flowers and in all flower organs: whorl1 tepals, whorl2 tepals, stamens and carpels, but not in leaves. Expression of CsatSEP3a/b/c/c_as cDNAs was compared in wild-type and mutant flowers. Expression of the isolatedCsatSEP3-like genes in whorl1 tepals together with E-class CsatAP1/FUL subfamily and B-class CsatAP3 and CsatPI subfamilies of genes, fits the ABCE “quartet model,” an extended form of the original ABC model proposed to explain the homeotic transformation of whorl1 sepals into whorl1 tepals in Liliales and Asparagales plants such as C. sativus. This conclusion was also supported by the interaction of the CsatSEP3b protein with CsatAP1/FUL and CsatAP3 proteins. In contrast, expression of both B-class CsatAP3 and CsatPI genes and the C-class CsatAGAMOUS genes together with E-class CsatSEP3-like genes in carpels, without any phenotypic effects on carpels, raises questions about the role of these gene classes in carpel formation in this non-grass monocot and requires further experimentation. Finally, taking advantage of the size and sequence differences in amplified genomic sequences of the triploid C. sativus and comparing them with the respective sequences from C. tomasii, C. hadriaticus and C. cartwrightianus, three putative wild-type diploid progenitor species, we examined the origin of CsatSEP3a sequence.     

Habitat type richness associations with environmental variables: a case study in the Greek Natura 2000 aquatic ecosystems

We investigated the potential associations of habitat type richness patterns with a series of environmental variables in 61 protected aquatic ecosystems of the Greek Natura 2000 network. Habitat type classification followed the Natura 2000 classification scheme. Habitat type richness was measured as the number of different habitat types in an area. To overcome a potential area effect in quantifying habitat type richness, we applied the “moving window” technique. The environmental variables were selected to account for some of the major threats to biodiversity, such as fragmentation, habitat loss and climate change. We run GLMs to associate habitat type richness with different combinations of climatic, spatial and topographic variables. Habitat type richness seemed to significantly associate with climatic variables, more than spatial or topographic ones. In particular, for the climatic ones, the importance of precipitation surpassed that of temperature and especially the precipitation of the wettest and driest month had a limiting contribution to richness unlike average climate estimators. Moreover, the landscape’s latitude and longitude and fragmentation were significantly associated to richness. Our findings are in accordance to those observed in recent literature at lower (i.e. species) levels of ecological organization, fact showing that large-scale phenomena (such as climate change) can also be observed at the habitat type level, at least in our case. Thus, following the context of the Habitats Directive (92/43/EEC), that habitat types and not solely species of community interest should be protected and restored, this study serves as a first step towards investigating habitat type richness patterns.     

Response of rigor cross-bridges to stretch detected by fluorescence lifetime imaging microscopy of myosin essential light chain in skeletal muscle fibers

We applied fluorescence lifetime imaging microscopy to map the microenvironment of the myosin essential light chain (ELC) in permeabilized skeletal muscle fibers. Four ELC mutants containing a single cysteine residue at different positions in the C-terminal half of the protein (ELC-127, ELC-142, ELC-160, and ELC-180) were generated by site-directed mutagenesis, labeled with 7-diethylamino-3-((((2-iodoacetamido)ethyl)amino)carbonyl)coumarin, and introduced into permeabilized rabbit psoas fibers. Binding to the myosin heavy chain was associated with a large conformational change in the ELC. When the fibers were moved from relaxation to rigor, the fluorescence lifetime increased for all label positions. However, when 1% stretch was applied to the rigor fibers, the lifetime decreased for ELC-127 and ELC-180 but did not change for ELC-142 and ELC-160. The differential change of fluorescence lifetime demonstrates the shift in position of the C-terminal domain of ELC with respect to the heavy chain and reveals specific locations in the lever arm region sensitive to the mechanical strain propagating from the actin-binding site to the lever arm.     

A biomolecule friendly photolithographic process for fabrication of protein microarrays on polymeric films coated on silicon chips

The last years, there is a steadily growing demand for methods and materials appropriate to create patterns of biomolecules for bioanalytical applications. Here, a photolithographic method for patterning biomolecules onto a silicon surface coated with a polymeric layer of high protein binding capacity is presented. The patterning process does not affect the polymeric film and the activity of the immobilized onto the surface biomolecules. Therefore, it permits sequential immobilization of different biomolecules on spatially distinct areas on the same solid support. The polymeric layer is based on a commercially available photoresist (AZ5214) that is cured at high temperature in order to provide a stable substrate for creation of protein microarrays by the developed photolithographic process. The photolithographic material consists of a (meth)acrylate copolymer and a sulfonium salt as a photoacid generator, and it is lithographically processed by thermal treatment at temperatures 相似文献   

Polycystin‐1 affects cancer cell behaviour and interacts with mTOR and Jak signalling pathways in cancer cell lines

Polycystic Kidney Disease (PKD), which is attributable to mutations in the PKD1 and PKD2 genes encoding polycystin‐1 (PC1) and polycystin‐2 (PC2) respectively, shares common cellular defects with cancer, such as uncontrolled cell proliferation, abnormal differentiation and increased apoptosis. Interestingly, PC1 regulates many signalling pathways including Jak/STAT, mTOR, Wnt, AP‐1 and calcineurin‐NFAT which are also used by cancer cells for sending signals that will allow them to acquire and maintain malignant phenotypes. Nevertheless, the molecular relationship between polycystins and cancer is unknown. In this study, we investigated the role of PC1 in cancer biology using glioblastoma (GOS3), prostate (PC3), breast (MCF7), lung (A549) and colorectal (HT29) cancer cell lines. Our in vitro results propose that PC1 promotes cell migration in GOS3 cells and suppresses cell migration in A549 cells. In addition, PC1 enhances cell proliferation in GOS3 cells but inhibits it in MCF7, A549 and HT29 cells. We also found that PC1 up‐regulates mTOR signalling and down‐regulates Jak signalling in GOS3 cells, while it up‐regulates mTOR signalling in PC3 and HT29 cells. Together, our study suggests that PC1 modulates cell proliferation and migration and interacts with mTOR and Jak signalling pathways in different cancer cell lines. Understanding the molecular details of how polycystins are associated with cancer may lead to the identification of new players in this devastating disease.