3-D Ultrastructure of O. tauri: Electron Cryotomography of an Entire Eukaryotic Cell

The hallmark of eukaryotic cells is their segregation of key biological functions into discrete, membrane-bound organelles. Creating accurate models of their ultrastructural complexity has been difficult in part because of the limited resolution of light microscopy and the artifact-prone nature of conventional electron microscopy. Here we explored the potential of the emerging technology electron cryotomography to produce three-dimensional images of an entire eukaryotic cell in a near-native state. Ostreococcus tauri was chosen as the specimen because as a unicellular picoplankton with just one copy of each organelle, it is the smallest known eukaryote and was therefore likely to yield the highest resolution images. Whole cells were imaged at various stages of the cell cycle, yielding 3-D reconstructions of complete chloroplasts, mitochondria, endoplasmic reticula, Golgi bodies, peroxisomes, microtubules, and putative ribosome distributions in-situ. Surprisingly, the nucleus was seen to open long before mitosis, and while one microtubule (or two in some predivisional cells) was consistently present, no mitotic spindle was ever observed, prompting speculation that a single microtubule might be sufficient to segregate multiple chromosomes

Impact of bleeding-related complications and/or blood product transfusions on hospital costs in inpatient surgical patients

<p>Abstract</p> <p>Background</p> <p>Inadequate surgical hemostasis may lead to transfusion and/or other bleeding-related complications. This study examines the incidence and costs of bleeding-related complications and/or blood product transfusions occurring as a consequence of surgery in various inpatient surgical cohorts.</p> <p>Methods</p> <p>A retrospective analysis was conducted using Premier's Perspective™ hospital database. Patients who had an inpatient procedure within a specialty of interest (cardiac, vascular, non-cardiac thoracic, solid organ, general, reproductive organ, knee/hip replacement, or spinal surgery) during 2006-2007 were identified. For each specialty, the rate of bleeding-related complications (including bleeding event, intervention to control for bleeding, and blood product transfusions) was examined, and hospital costs and length of stay (LOS) were compared between surgeries with and without bleeding-related complications. Incremental costs and ratios of average total hospital costs for patients with bleeding-related complications vs. those without complications were estimated using ordinary least squares (OLS) regression, adjusting for demographics, hospital characteristics, and other baseline characteristics. Models using generalized estimating equations (GEE) were also used to measure the impact of bleeding-related complications on costs while accounting for the effects related to the clustering of patients receiving care from the same hospitals.</p> <p>Results</p> <p>A total of 103,829 cardiac, 216,199 vascular, 142,562 non-cardiac thoracic, 45,687 solid organ, 362,512 general, 384,132 reproductive organ, 246,815 knee/hip replacement, and 107,187 spinal surgeries were identified. Overall, the rate of bleeding-related complications was 29.9% and ranged from 7.5% to 47.4% for reproductive organ and cardiac, respectively. Overall, incremental LOS associated with bleeding-related complications or transfusions (unadjusted for covariates) was 6.0 days and ranged from 1.3 to 9.6 days for knee/hip replacement and non-cardiac thoracic, respectively. The incremental cost per hospitalization associated with bleeding-related complications and adjusted for covariates was highest for spinal surgery ($17,279) followed by vascular ($15,123), solid organ ($13,210), non-cardiac thoracic ($13,473), cardiac ($10,279), general ($4,354), knee/hip replacement ($3,005), and reproductive organ ($2,805).</p> <p>Conclusions</p> <p>This study characterizes the increased hospital LOS and cost associated with bleeding-related complications and/or transfusions occurring as a consequence of surgery, and supports implementation of blood-conservation strategies.</p

Structural Diversity in Bacterial Ribosomes: Mycobacterial 70S Ribosome Structure Reveals Novel Features

Here we present analysis of a 3D cryo-EM map of the 70S ribosome from Mycobacterium smegmatis, a saprophytic cousin of the etiological agent of tuberculosis in humans, Mycobacterium tuberculosis. In comparison with the 3D structures of other prokaryotic ribosomes, the density map of the M. smegmatis 70S ribosome reveals unique structural features and their relative orientations in the ribosome. Dramatic changes in the periphery due to additional rRNA segments and extra domains of some of the peripheral ribosomal proteins like S3, S5, S16, L17, L25, are evident. One of the most notable features appears in the large subunit near L1 stalk as a long helical structure next to helix 54 of the 23S rRNA. The sharp upper end of this structure is located in the vicinity of the mRNA exit channel. Although the M. smegmatis 70S ribosome possesses conserved core structure of bacterial ribosome, the new structural features, unveiled in this study, demonstrates diversity in the 3D architecture of bacterial ribosomes. We postulate that the prominent helical structure related to the 23S rRNA actively participates in the mechanisms of translation in mycobacteria

Optimization of Energy-Consuming Pathways towards Rapid Growth in HPV-Transformed Cells

Cancer is a complex, multi-step process characterized by misregulated signal transduction and altered metabolism. Cancer cells divide faster than normal cells and their growth rates have been reported to correlate with increased metabolic flux during cell transformation. Here we report on progressive changes in essential elements of the biochemical network, in an in vitro model of transformation, consisting of primary human keratinocytes, human keratinocytes immortalized by human papillomavirus 16 (HPV16) and passaged repeatedly in vitro, and the extensively-passaged cells subsequently treated with the carcinogen benzo[a]pyrene. We monitored changes in cell growth, cell size and energy metabolism. The more transformed cells were smaller and divided faster, but the cellular energy flux was unchanged. During cell transformation the protein synthesis network contracted, as shown by the reduction in key cap-dependent translation factors. Moreover, there was a progressive shift towards internal ribosome entry site (IRES)-dependent translation. The switch from cap to IRES-dependent translation correlated with progressive activation of c-Src, an activator of AMP-activated protein kinase (AMPK), which controls energy-consuming processes, including protein translation. As cellular protein synthesis is a major energy-consuming process, we propose that the reduction in cell size and protein amount provide energy required for cell survival and proliferation. The cap to IRES-dependent switch seems to be part of a gradual optimization of energy-consuming mechanisms that redirects cellular processes to enhance cell growth, in the course of transformation

Comparative evaluation of the tendon-bone interface contact pressure in different single- versus double-row suture anchor repair techniques

The aim of the study was to evaluate the time zero contact pressure over a defined rotator cuff footprint using different repair and stitch techniques in an established sheep model. Forty fresh-frozen sheep shoulders were randomly assigned to five repair groups: single-row repair using simple stitches (SRA-s), single-row repair using horizontal mattress stitches (SRA-m), and single-row repair using arthroscopic Mason-Allen stitches (SRA-ama). Double-row repair was either performed with a combination of simple and horizontal mattress stitches (DRA-sm) or with arthroscopic Mason-Allen/horizontal mattress stitches (DRA-amam). Investigations were performed using a pressure-sensitive film system. The average contact pressure and pressure pattern were measured for each group. Contact pressure was lowest in SRA-m followed by SRA-s. SRA-ama showed highest contact pressure of all single-row treatment groups (P < 0.05). DRA-amam presented the highest overall contact pressure (P < 0.05), whereas DRA-sm exerted contact pressure equal to that of SRA-ama. Both double-row techniques showed the most expanded pressure pattern. Average contact pressures for the more complex single- and double-row techniques utilizing arthroscopic Mason-Allen stitches were greater than were those of the repair techniques utilizing simple and horizontal mattress stitches. However, the contact pattern between the anchors could be increased by using the double-row technique, resulting in more footprint coverage compared to patterns utilizing the single-row techniques. These results support the use of the more complex arthroscopic Mason-Allen stitches and may improve the environment for healing of the repaired rotator cuff tendon

Protein-Protein Interactions within Late Pre-40S Ribosomes

Ribosome assembly in eukaryotic organisms requires more than 200 assembly factors to facilitate and coordinate rRNA transcription, processing, and folding with the binding of the ribosomal proteins. Many of these assembly factors bind and dissociate at defined times giving rise to discrete assembly intermediates, some of which have been partially characterized with regards to their protein and RNA composition. Here, we have analyzed the protein-protein interactions between the seven assembly factors bound to late cytoplasmic pre-40S ribosomes using recombinant proteins in binding assays. Our data show that these factors form two modules: one comprising Enp1 and the export adaptor Ltv1 near the beak structure, and the second comprising the kinase Rio2, the nuclease Nob1, and a regulatory RNA binding protein Dim2/Pno1 on the front of the head. The GTPase-like Tsr1 and the universally conserved methylase Dim1 are also peripherally connected to this second module. Additionally, in an effort to further define the locations for these essential proteins, we have analyzed the interactions between these assembly factors and six ribosomal proteins: Rps0, Rps3, Rps5, Rps14, Rps15 and Rps29. Together, these results and previous RNA-protein crosslinking data allow us to propose a model for the binding sites of these seven assembly factors. Furthermore, our data show that the essential kinase Rio2 is located at the center of the pre-ribosomal particle and interacts, directly or indirectly, with every other assembly factor, as well as three ribosomal proteins required for cytoplasmic 40S maturation. These data suggest that Rio2 could play a central role in regulating cytoplasmic maturation steps

Tendon–bone contact pressure and biomechanical evaluation of a modified suture-bridge technique for rotator cuff repair

The aim of the study was to evaluate the time-zero mechanical and footprint properties of a suture-bridge technique for rotator cuff repair in an animal model. Thirty fresh-frozen sheep shoulders were randomly assigned among three investigation groups: (1) cyclic loading, (2) load-to-failure testing, and (3) tendon–bone interface contact pressure measurement. Shoulders were cyclically loaded from 10 to 180 N and displacement to gap formation of 5- and 10-mm at the repair site. Cycles to failure were determined. Additionally, the ultimate tensile strength and stiffness were verified along with the mode of failure. The average contact pressure and pressure pattern were investigated using a pressure-sensitive film system. All of the specimens resisted against 3,000 cycles and none of them reached a gap formation of 10 mm. The number of cycles to 5-mm gap formation was 2,884.5 ± 96.8 cycles. The ultimate tensile strength was 565.8 ± 17.8 N and stiffness was 173.7 ± 9.9 N/mm. The entire specimen presented a unique mode of failure as it is well known in using high strength sutures by pulling them through the tendon. We observed a mean contact pressure of 1.19 ± 0.03 MPa, applied on the footprint area. The fundamental results of our study support the use of a suture-bridge technique for optimising the conditions of the healing biology of a reconstructed rotator cuff tendon. Nevertheless, an individual estimation has to be done if using the suture-bridge technique clinically. Further investigation is necessary to evaluate the cell biological healing process in order to achieve further sufficient advancements in rotator cuff repair