Editing activity for eliminating mischarged tRNAs is essential in mammalian mitochondria

Accuracy of protein synthesis is enabled by the selection of amino acids for tRNA charging by aminoacyl-tRNA synthetases (ARSs), and further enhanced by the proofreading functions of some of these enzymes for eliminating tRNAs mischarged with noncognate amino acids. Mouse models of editing-defective cytoplasmic alanyl-tRNA synthetase (AlaRS) have previously demonstrated the importance of proofreading for cytoplasmic protein synthesis, with embryonic lethal and progressive neurodegeneration phenotypes. Mammalian mitochondria import their own set of nuclear-encoded ARSs for translating critical polypeptides of the oxidative phosphorylation system, but the importance of editing by the mitochondrial ARSs for mitochondrial proteostasis has not been known. We demonstrate here that the human mitochondrial AlaRS is capable of editing mischarged tRNAs in vitro, and that loss of the proofreading activity causes embryonic lethality in mice. These results indicate that tRNA proofreading is essential in mammalian mitochondria, and cannot be overcome by other quality control mechanisms

An engineered mammalian band-pass network

Gene expression circuitries, which enable cells to detect precise levels within a morphogen concentration gradient, have a pivotal impact on biological processes such as embryonic pattern formation, paracrine and autocrine signalling, and cellular migration. We present the rational synthesis of a synthetic genetic circuit exhibiting band-pass detection characteristics. The components, involving multiply linked mammalian trans-activator and -repressor control systems, were selected and fine-tuned to enable the detection of ‘low-threshold’ morphogen (tetracycline) concentrations, in which target gene expression was triggered, and a ‘high-threshold’ concentration, in which expression was muted. In silico predictions and supporting experimental findings indicated that the key criterion for functional band-pass detection was the matching of componentry that enabled sufficient separation of the low and high threshold points. Using the circuitry together with a fluorescence-encoded target gene, mammalian cells were genetically engineered to be capable of forming a band-like pattern of differentiation in response to a tetracycline chemical gradient. Synthetic gene networks designed to emulate naturally occurring gene behaviours provide not only insight into biological processes, but may also foster progress in future tissue engineering, gene therapy and biosensing applications

A retrospective analysis of bilateral fractures over sixteen years: localisation and variation in treatment of second hip fractures

The aim of this study was the evaluation of contralateral hip fractures after a previous hip fracture. For this retrospective analysis patients were selected from the database of the LUMC, a teaching hospital in the south-west of the Netherlands. We analyzed all patients with a second fracture of a hip between 1992 and 2007. The exclusion criteria were high impact trauma and patients with diseases or medication known to have a negative effect on bone metabolism. A total of 1,604 hip fractures were identified. The possible predictive factors for the second fracture and descriptive statistics related to surgery (Hb and HT before and after the operation, total amount of intra- and postoperative blood loss, type of osteosynthesis, complications, time of death after the last fracture, time between arrival in the hospital and operation and hospital stay for both fractures) were recorded. A total of 32 second hip fractures were identified (2%) at a mean of 27.5 (SD 28.9) months after the initial hip fracture. The mean age at the first fracture was 77.2 years (SD 11.7), and 27 of 32 patients were female. Of these 32 patients (64 bilateral hip fractures), 32 fractures were intracapsular (1 femoral neck, 31 subcapital) and 32 were extracapsular fractures (6 subtrochanteric, 26 transtrochanteric). Although 24 of the 32 patients had identical first and second hip fractures, only eight out of 32 hips were treated with the same implants. There was a significant difference in Singh index between both hips at the time of the first fracture. There was also a significant difference in Singh index between the hip which was not fractured compared with its subsequent index when it was broken. All other studied patient and fracture characteristics were not significantly different. In this population the percentage of second hip fractures was relatively low compared to other studies. The choice of implants in this study shows that implants were chosen randomly. Because there is a significant difference in the Singh index during first and second hip fracture, osteoporosis medication might help reduce the incidence of second hip fractures

The effect of time-to-surgery on outcome in elderly patients with proximal femoral fractures

<p>Abstract</p> <p>Background</p> <p>Whether reducing time-to-surgery for elderly patients suffering from hip fracture results in better outcomes remains subject to controversial debates.</p> <p>Methods</p> <p>As part of a prospective observational study conducted between January 2002 and September 2003 on hip-fracture patients from 268 acute-care hospitals all over Germany, we investigated the relationship of time-to-surgery with frequency of post-operative complications and one-year mortality in elderly patients (age ≥65) with isolated proximal femoral fracture (femoral neck fracture or pertrochanteric femoral fracture). Patients with short (≤12 h), medium (> 12 h to ≤36 h) and long (> 36 h) times-to-surgery, counting from the time of the fracture event, were compared for patient characteristics, operative procedures, post-operative complications and one-year mortality.</p> <p>Results</p> <p>Hospital data were available for 2916 hip-fracture patients (mean age (SD) in years: 82.1 (7.4), median age: 82; 79.7% women). Comparison of groups with short (n = 802), medium (n = 1191) and long (n = 923) time-to-surgery revealed statistically significant differences in a few patient characteristics (age, American Society of Anesthesiologists ratings classification and type of admission) and in operative procedures (total hip endoprosthesis, hemi-endoprosthetic implants, other osteosynthetic procedures). However, comparison of these same groups for frequency of postoperative complications revealed only some non-significant associations with certain complications such as post-operative bleeding requiring treatment (early surgery patients) and urinary tract infections (delayed surgery patients). Both unadjusted rates of one-year all-cause mortality (between 18.1% and 20.5%), and the multivariate-adjusted hazard ratios (HR for time-to-surgery: 1.04; p = 0.55) showed no association between mortality and time-to-surgery.</p> <p>Conclusion</p> <p>Although this study found a trend toward more frequent post-operative complications in the longest time-to-surgery group, there was no effect of time-to-surgery on mortality. Shorter time-to-surgery may be associated with somewhat lower rates of post-operative complications such as decubitus ulcers, urinary tract infections, thromboses, pneumonia and cardiovascular events, and with somewhat higher rates of others such as post-operative bleeding or implant complications.</p

Ectopic Wnt/Beta–Catenin Signaling Induces Neurogenesis in the Spinal Cord and Hindbrain Floor Plate

The most ventral structure of the developing neural tube, the floor plate (FP), differs in neurogenic capacity along the neuraxis. The FP is largely non-neurogenic at the hindbrain and spinal cord levels, but generates large numbers of dopamine (mDA) neurons at the midbrain levels. Wnt1, and other Wnts are expressed in the ventral midbrain, and Wnt/beta catenin signaling can at least in part account for the difference in neurogenic capacity of the FP between midbrain and hindbrain levels. To further develop the hypothesis that canonical Wnt signaling promotes mDA specification and FP neurogenesis, we have generated a model wherein beta–catenin is conditionally stabilized throughout the FP. Here, we unambiguously show by fate mapping FP cells in this mutant, that the hindbrain and spinal cord FP are rendered highly neurogenic, producing large numbers of neurons. We reveal that a neurogenic hindbrain FP results in the altered settling pattern of neighboring precerebellar neuronal clusters. Moreover, in this mutant, mDA progenitor markers are induced throughout the rostrocaudal axis of the hindbrain FP, although TH+ mDA neurons are produced only in the rostral aspect of rhombomere (r)1. This is, at least in part, due to depressed Lmx1b levels by Wnt/beta catenin signaling; indeed, when Lmx1b levels are restored in this mutant, mDA are observed not only in rostral r1, but also at more caudal axial levels in the hindbrain, but not in the spinal cord. Taken together, these data elucidate both patterning and neurogenic functions of Wnt/beta catenin signaling in the FP, and thereby add to our understanding of the molecular logic of mDA specification and neurogenesis

COX-2-mediated stimulation of the lymphangiogenic factor VEGF-C in human breast cancer

Increased expression of COX-2 or VEGF-C has been correlated with progressive disease in certain cancers. Present study utilized several human breast cancer cell lines (MCF-7, T-47D, Hs578T and MDA-MB-231, varying in COX-2 expression) as well as 10 human breast cancer specimens to examine the roles of COX-2 and prostaglandin E (EP) receptors in VEGF-C expression or secretion, and the relationship of COX-2 or VEGF-C expression to lymphangiogenesis. We found a strong correlation between COX-2 mRNA expression and VEGF-C expression or secretion levels in breast cancer cell lines and VEGF-C expression in breast cancer tissues. Expression of LYVE-1, a selective marker for lymphatic endothelium, was also positively correlated with COX-2 or VEGF-C expression in breast cancer tissues. Inhibition of VEGF-C expression and secretion in the presence of COX-1/2 or COX-2 inhibitors or following downregulation of COX-2 with COX-2 siRNA established a stimulatory role COX-2 in VEGF-C synthesis by breast cancer cells. EP1 as well as EP4 receptor antagonists inhibited VEGF-C production indicating the roles of EP1 and EP4 in VEGF-C upregulation by endogenous PGE2. Finally, VEGF-C secretion by MDA-MB-231 cells was inhibited in the presence of kinase inhibitors for Her-2/neu, Src and p38 MAPK, indicating a requirement of these kinases for VEGF-C synthesis. These results, for the first time, demonstrate a regulatory role of COX-2 in VEGF-C synthesis (and thereby lymphangiogenesis) in human breast cancer, which is mediated at least in part by EP1/EP4 receptors

A High-Throughput Platform for Lentiviral Overexpression Screening of the Human ORFeome

In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2′-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes

Identification of a novel proliferation-inducing determinant using lentiviral expression cloning

One of the major challenges in the post‐genome era is the correlation between genes and function or phenotype. We have pioneered a strategy for screening of cDNA libraries, which is based on sequential combination of lentiviral and oncoretroviral expression systems and can be used to identify proliferation‐modulating genes. Screening of a lentiviral expression library derived from adult human brain cDNA resulted in cloning of the potent proliferation‐inducing determinant termed pi1 (proliferation inducer 1). Transduction experiments using GFP‐expressing oncoretroviruses to target proliferation‐competent cells suggested that overexpression of pi1 initiates proliferation of human umbilical vein endothelial cells (HUVECs). Growth induction of HUVECs as well as Swiss3T3 fibroblasts was confirmed by Brd‐uridine incorporation assays, which correlated increased DNA synthesis with expression of pi1. The identified pi1 cDNA is 297 bp long and encodes a 10 kDa polypeptide. Since deregulation of proliferation control accounts for a number of today's untreatable human diseases such as neurodegenerative disorders and cancer, discovery of novel proliferation‐modulating genes is essential for developing new strategies for gene therapy and tissue engineerin