In vitro and in vivo single-molecule fluorescence imaging of ribosome-catalyzed protein synthesis

Combined with the availability of highly purified, fluorescently labeled in vitro translation systems, the advent of single-molecule fluorescence imaging has ushered in a new era in high-resolution mechanistic studies of ribosome-catalyzed protein synthesis, or translation. Together with ensemble biochemical investigations of translation and structural studies of functional ribosomal complexes, in vitro single-molecule fluorescence imaging of protein synthesis is providing unique mechanistic insight into this fundamental biological process. More recently, rapidly evolving breakthroughs in fluorescence-based molecular imaging in live cells with sub-diffraction-limit spatial resolution and ever-increasing temporal resolution provide great promise for conducting mechanistic studies of translation and its regulation in living cells. Here we review the remarkable recent progress that has been made in these fields, highlight important mechanistic insights that have been gleaned from these studies thus far, and discuss what we envision lies ahead as these approaches continue to evolve and expand to address increasingly complex mechanistic and regulatory aspects of translation.     

Early evolutionary differentiation of morphological variation in the mandible of South American caviomorph rodents (Rodentia, Caviomorpha)

Caviomorphs are a clade of South American rodents recorded at least since the early Oligocene (> 31.5 Ma) that exhibit ample eco-morphological variation. It has been proposed that phylogenetic structure is more important than ecological factors for understanding mandibular shape variation in this clade. This was interpreted as a result of the long-standing evolutionary history of caviomorphs and the early divergence of major lineages. In this work, we test this hypothesis through the analysis of morphological variation in the mandible of living and extinct species and compare this information with that obtained through comparative phylogenetic analyses. Our results support the hypothesis of early origin of mandibular variation; moreover, they suggest the conservation of early differentiated morphologies, which could indicate the existence of constrained evolutionary diversification.     

Polarized migration of lymphatic endothelial cells is critically dependent on podoplanin regulation of Cdc42

We have shown previously that T1α/podoplanin is required for capillary tube formation by human lung microvascular lymphatic endothelial cells (HMVEC-LLy) and that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA shortly after the beginning of the lymphangiogenic process. The objective of this study was to determine whether podoplanin regulates HMVEC-LLy migration and whether this regulation is via modulation of small GTPase activation. In analysis of scratch wound assays, we found that small interfering RNA (siRNA) depletion of podoplanin expression in HMVEC-LLy inhibits VEGF-induced microtubule-organizing center (MTOC) and Golgi polarization and causes a dramatic reduction in directional migration compared with control siRNA-transfected cells. In addition, a striking redistribution of cortical actin to fiber networks across the cell body is observed in these cells, and, remarkably, it returns to control levels if the cells are cotransfected with a dominant-negative mutant of Cdc42. Moreover, cotransfection of a dominant-negative construct of Cdc42 into podoplanin knockdown HMVEC-LLy completely abrogated the effect of podoplanin deficiency, rescuing MTOC and Golgi polarization and cell migration to control level. Importantly, expression of constitutively active Cdc42 construct, like podoplanin knockdown, decreased RhoA-GTP level in HMVEC-LLy, demonstrating cross talk between both GTPases. Taken together, the results indicate that polarized migration of lymphatic endothelial cells in response to VEGF is mediated via a pathway of podoplanin regulation of small GTPase activities, in particular Cdc42.     

Identification of molecular-mimicry-based ligands for cholera diagnostics using magnetic relaxation

When covalently bound to an appropriate ligand, iron oxide nanoparticles can bind to a specific target of interest. This interaction can be detected through changes in the solution's spin-spin relaxation times (T2) via magnetic relaxation measurements. In this report, a strategy of molecular mimicry was used in order to identify targeting ligands that bind to the cholera toxin B subunit (CTB). The cellular CTB-receptor, ganglioside GM1, contains a pentasaccharide moiety consisting in part of galactose and glucose units. We therefore predicted that CTB would recognize carbohydrate-conjugated iron oxide nanoparticles as GM1 mimics, thus producing a detectable change in the T2 relaxation times. Magnetic relaxation experiments demonstrated that CTB interacted with the galactose-conjugated nanoparticles. This interaction was confirmed via surface plasmon resonance studies using either the free or nanoparticle-conjugated galactose molecule. The galactose-conjugated nanoparticles were then used as CTB sensors achieving a detection limit of 40 pM. Via magnetic relaxation studies, we found that CTB also interacted with dextran-coated nanoparticles, and surface plasmon resonance studies also confirmed this interaction. Additional experiments demonstrated that the dextran-coated nanoparticle can also be used as CTB sensors and that dextran can prevent the internalization of CTB into GM1-expressing cells. Our work indicates that magnetic nanoparticle conjugates and magnetic relaxation detection can be used as a simple and fast method to identify targeting ligands via molecular mimicry. Furthermore, our results show that the dextran-coated nanoparticles represent a low-cost approach for CTB detection.     

Age-related instability in spermatogenic cell nuclear and mitochondrial DNA obtained from Apex1 heterozygous mice

The prevalence of spontaneous mutations increases with age in the male germline; consequently, older men have an increased risk of siring children with genetic disease due to de novo mutations. The lacI transgenic mouse can be used to study paternal age effects, and in this system, the prevalence of de novo mutations increases in the male germline at old ages. Mutagenesis is linked with DNA repair capacity, and base excision repair (BER), which can ameliorate spontaneous DNA damage, decreases in nuclear extracts of spermatogenic cells from old mice. Mice heterozygous for a null allele of the Apex1 gene, which encodes apurinic/apyrimidinic endonuclease I (APEN), an essential BER enzyme, display an accelerated increase in spontaneous germline mutagenesis early in life. Here, the consequences of lifelong reduction of APEN on genetic instability in the male germline were examined, for the first time, at middle and old ages. Mutant frequency increased earlier in spermatogenic cells from Apex1(+/-) mice (by 6 months of age). Nuclear DNA damage increased with age in the spermatogenic lineage for both wild-type and Apex1(+/-) mice. By old age, mutant frequencies were similar for wild-type and APEN-deficient mice. Mitochondrial genome repair also depends on APEN, and novel analysis of mitochondrial DNA (mtDNA) damage revealed an increase in the Apex1(+/-) spermatogenic cells by middle age. Thus, Apex1 heterozygosity results in accelerated damage to mtDNA and spontaneous mutagenesis, consistent with an essential role for APEN in maintaining nuclear and mtDNA integrity in spermatogenic cells throughout life.     

Myeloablative therapy against high risk Ewing's sarcoma: A single institution experience and literature review

Background

Attempts to improve survival outcomes of patients with high risk Ewing''s sarcoma (ES) have focused on chemotherapy dose intensification strategies.

Aim

The objective of this study is to retrospectively evaluate clinical characteristics and outcome of pediatric patients with high risk ES treated at a single institution.

Materials and methods

From 1995 to 2008, seventeen patients (male:female, 14:3) were treated with dose-intensive therapy in our institution. Median age at diagnosis was 10 years (range: 2–15). Seven patients had metastases at diagnosis (lung in 6 cases and bone in one case). Eleven patients presented with unresectable disease. Fifteen (88.2%) received the Spanish Society of Pediatric Oncology protocol which includes six cycles of vincristine, doxorubicin, ifosfamide and etoposide. Two out of the six cases that were resectable received postoperative radiation. In addition, eleven patients received definitive radiation therapy. Finally, twelve (70.5%) out of 17 patients received myeloablative therapy with melphalan/etoposide. The rest of patients (N = 5) received busulfan/melphalan.

Results

Median follow-up was 78 months (range: 15–155 months). Initial responses were complete in all patients, but 9 of them developed progression disease. Seven patients became long-term event-free survivors. No patient died of toxicity after transplantation. The 2- and 5-year overall survival rates for all patients were 93% and 73%, respectively. Event-free survival rates were 74% and 54% at 2 and 5 years, respectively.

Conclusion

This single-institution experience suggests that myeloablative therapy against high risk ES is effective and safe.     

Hsp90 governs dispersion and drug resistance of fungal biofilms

Fungal biofilms are a major cause of human mortality and are recalcitrant to most treatments due to intrinsic drug resistance. These complex communities of multiple cell types form on indwelling medical devices and their eradication often requires surgical removal of infected devices. Here we implicate the molecular chaperone Hsp90 as a key regulator of biofilm dispersion and drug resistance. We previously established that in the leading human fungal pathogen, Candida albicans, Hsp90 enables the emergence and maintenance of drug resistance in planktonic conditions by stabilizing the protein phosphatase calcineurin and MAPK Mkc1. Hsp90 also regulates temperature-dependent C. albicans morphogenesis through repression of cAMP-PKA signalling. Here we demonstrate that genetic depletion of Hsp90 reduced C. albicans biofilm growth and maturation in vitro and impaired dispersal of biofilm cells. Further, compromising Hsp90 function in vitro abrogated resistance of C. albicans biofilms to the most widely deployed class of antifungal drugs, the azoles. Depletion of Hsp90 led to reduction of calcineurin and Mkc1 in planktonic but not biofilm conditions, suggesting that Hsp90 regulates drug resistance through different mechanisms in these distinct cellular states. Reduction of Hsp90 levels led to a marked decrease in matrix glucan levels, providing a compelling mechanism through which Hsp90 might regulate biofilm azole resistance. Impairment of Hsp90 function genetically or pharmacologically transformed fluconazole from ineffectual to highly effective in eradicating biofilms in a rat venous catheter infection model. Finally, inhibition of Hsp90 reduced resistance of biofilms of the most lethal mould, Aspergillus fumigatus, to the newest class of antifungals to reach the clinic, the echinocandins. Thus, we establish a novel mechanism regulating biofilm drug resistance and dispersion and that targeting Hsp90 provides a much-needed strategy for improving clinical outcome in the treatment of biofilm infections.     

Transgenic overexpression of PKCε in the mouse prostate induces preneoplastic lesions

It is well established that protein kinase C (PKC) isozymes play distinctive roles in mitogenic and survival signaling as well as in cancer progression. PKCε, the product of the PRKCE gene, is upregulated in various types of cancers including prostate, lung and breast cancer. To address a potential role for PKCs in prostate cancer progression we generated three mouse transgenic lines expressing PKCα, PKCδ or PKCε in the prostate epithelium under the control of the rat probasin (PB) promoter. Whereas PB-PKCα and PB-PKCδ mice did not show any evident phenotype, PB-PKCε mice developed prostate hyperplasia as well as prostate intraepithelial neoplasia (PIN) that displayed enhanced phospho-Akt, phospho-S6 and phospho-Stat3 levels, as well as enhanced resistance to apoptotic stimuli. PKCε overexpression was insufficient to drive neoplastic changes in the mouse prostate. Notably, overexpression of PKCε by adenoviral means in normal immortalized RWPE-1 prostate cells confers a growth advantage and hyperactivation of Erk and Akt. Our results argue for a causal link between PKCε overexpression and prostate cancer development.Key words: PKCε, transgenic mice, prostate, preneoplastic lesions, cell survival, Akt