Different modes of programmed cell death during oogenesis of the silkmoth Bombyx mori

It is increasingly recognized that programmed cell death includes not only apoptosis and autophagy, but also other types of nonapoptotic cell death, such as paraptosis, which are all characterized by distinct morphological features. Our findings indicate that all three types of programmed cell death occur in the ovarian nurse cell cluster during late vitellogenesis (formation of the egg yolk) of Bombyx mori (Lepidoptera), whereas middle vitellogenesis is exclusively characterized by the presence of a nonapoptotic type of cell death, known as paraptosis. During middle vitellogenesis, nurse cells exhibit clearly cytoplasmic vacuolization, as revealed by ultrastructural examination performed through conventional light and transmission electron microscopy, while no signs of apoptotic or autophagic features are detectable. Moreover, nurse cells of developmental stages 7, 8 and 9 contain autophagic compartments, as well as apoptotic characteristics, such as condensed chromatin, fragmented DNA and activated caspases, as revealed by in vitro assays. We propose that paraptosis precedes both apoptosis and autophagy during vitellogenesis, since its initial activation is detectable during middle vitellogenesis, whereas no apoptotic nor autophagic features are observed. In contrast, at the late stages of Bombyx mori oogenesis, paraptosis, autophagy and apoptosis operate synergistically, resulting in a more efficient elimination of the degenerated nurse cells.     

Increases in serum concentration of human heart-type fatty acid-binding protein following elective coronary intervention

Background: Heart-type fatty acid-binding protein (H-FABP) is considered a marker of myocardial necrosis but whether or not it is modified by myocardial ischemia is not clear. We sought to investigate if H-FABP serum levels increase following non-urgent coronary angioplasty.

Methods: We studied 31 patients undergoing coronary angioplasty. Peripheral venous samples were drawn immediately before angioplasty, 1?h after the first balloon inflation and 24?h after the procedure and assayed for H-FABP.

Results: Serum levels of H-FABP increased significantly at 1?h vs baseline from 2554?±?1268 to 3322?±?245?pg?ml^?1 (p?=?0.024). However, no differences were observed between 1?h and 24?h after angioplasty (3268?±?1861 vs 3322?±?2459?pg ml^?1, p?=?0.87). Moreover, no significant difference was observed when we compared 24?h after angioplasty with the baseline (3268?±?1861vs 2554?±?1268?pg ml^?1, p?=?0.112).

Conclusions: We conclude that H-FABP significantly increases after elective coronary angioplasty at 1?h compared with baseline values; whether or not this has any prognostic significance for future events, as it occurs with troponins, needs to be studied further.     

A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis

Background

The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined.

Results

We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called “RodCellJ”, allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. (Continued on next page)(Continued from previous page)

Conclusions

“RodCellJ” is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells.

Availability

RodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html.

     

Neuromuscular efficiency during sit to stand movement in women with knee osteoarthritis

The purpose of this study was to investigate the neuromuscular efficiency of women with knee osteoarthritis (OA) when performing a sit-to-stand movement and during maximum strength efforts. Twelve women with unilateral knee OA (age 60.33 ± 6.66 years, height 1.61 ± 0.05 m, mass 77.08 ± 9.2 kg) and 11 controls (age 56.54 ± 5.46 years, height 1.64 ± 0.05 m, mass 77.36 ± 13.34 kg) participated in this study. Subjects performed a sit-to-stand movement from a chair while position of center of pressure and knee angular speed were recorded. Furthermore, maximal isokinetic knee extension and flexion strength at 60°/s, 120°/s and 150°/s was measured. Surface, electromyography (EMG) from the biceps femoris (BF), vastus lateralis (VL) and vastus medialis (VM) was recorded during all tests. Analysis of variance (ANOVA) showed that during the sit-to-stand OA group demonstrated significantly lower knee angular speed (44.49 ± 9.61°/s vs. 71.68 ± 19.86°/s), a more posterior position of the center of pressure (39.20 ± 7.02% vs. 41.95 ± 2.49%) and a higher antagonist BF activation (57.13 ± 20.55% vs. 32.01 ± 19.5%) compared with controls (p < 0.05). Further, women with knee OA demonstrated a lower Moment-to-EMG ratio than controls in extension and eccentric flexion at 60°/s and 150°/s, while the opposite was found for concentric flexion at 60°/s (p < 0.05). Among other factors, the slower performance of the sit-to-stand movement in women with OA is due to a less efficient use of the knee extensor muscles (less force per unit of EMG) and, perhaps, a higher BF antagonist co-activation. This may lead subjects with OA to adopt a different movement strategy compared with controls.     

Inhibin secretion in women with the polycystic ovary syndrome before and after treatment with progesterone

Objectives  

It has been suggested that inhibin secretion is altered in women with the polycystic ovary syndrome (PCOS). However, the contribution of a preceding luteal phase has not been taken into account. The aim of the present study was to investigate whether progesterone in the context of a simulated luteal phase affects basal and FSH-induced inhibin secretion in women with PCOS and elevated LH.     

Glutamate Dehydrogenase Isoforms with N-Terminal (His)6- or FLAG-Tag Retain Their Kinetic Properties and Cellular Localization

Glutamate dehydrogenase (GDH) is a crucial enzyme on the crossroads of amino acid and energy metabolism and it is operating in all domains of life. According to current knowledge GDH is present only in one functional isoform in most animals, including mice. In addition to this housekeeping enzyme (hGDH1 in humans), humans and apes have acquired a second isoform (hGDH2) with a distinct tissue expression profile. In the current study we have cloned both mouse and human GDH constructs containing FLAG and (His)₆ small genetically-encoded tags, respectively. The hGDH1 and hGDH2 constructs containing N-terminal (His)₆ tags were successfully expressed in Sf9 cells and the recombinant proteins were isolated to ≥95 % purity in a two-step procedure involving ammonium sulfate precipitation and Ni²⁺-based immobilized metal ion affinity chromatography. To explore whether the presence of the FLAG and (His)₆ tags affects the cellular localization and functionality of the GDH isoforms, we studied the subcellular distribution of the expressed enzymes as well as their regulation by adenosine diphosphate monopotassium salt (ADP) and guanosine-5′-triphosphate sodium salt (GTP). Through immunoblot analysis of the mitochondrial and cytosolic fraction of the HEK cells expressing the recombinant proteins we found that neither FLAG nor (His)₆ tag disturbs the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial mouse GDH1 or human hGDH1 and hGDH2 did not change the ADP activation or GTP inhibition pattern of the proteins as compared to their untagged counterparts. However, the addition of FLAG tag to the C-terminus of the mouse GDH left the recombinant protein fivefold less sensitive to ADP activation. This finding highlights the necessity of the functional characterization of recombinant proteins containing even the smallest available tags.     

Combined Ablation and Resection (CARe) as an Effective Parenchymal Sparing Treatment for Extensive Colorectal Liver Metastases

Background

Combined intra-operative ablation and resection (CARe) is proposed to treat extensive colorectal liver metastases (CLM). This multicenter study was conducted to evaluate overall survival (OS), local recurrence-free survival (LRFS), hepatic recurrence-free survival (HRFS) and progression-free survival (PFS), to identify factors associated with survival, and to report complications.

Materials and Methods

Four centers combined retropectively their clinical experiences regarding CLM treated by CARe. CLM characteristics, pre- and post-operative chemotherapy regimens, surgical procedures, complications and survivals were analyzed.

Results

Of the 288 patients who received CARe, 210 (73%) had synchronous and 255 (88%) had bilateral CLM. Twenty-two patients (8%) had extrahepatic disease. Median follow-up was 3.17 years (95%CI 2.83–4.08). Median OS was 3.33 years (95%CI 3.08–4.17) and 5-year OS was 37% (95%CI 29–45). One- and 5-year LRFS from ablated lesions were 87.9% (95%CI 83.3–91.2) and 78.0% (95%CI 71–83), respectively. Median HRFS and PFS were 14 months (95%CI 11–18) and 9 months (95%CI 8–11), respectively. One hundred patients experienced complications: 29 grade I, 68 grade II–III–IV, and three deaths. In the multivariate models adjusted for center, the occurrence of complications was confirmed as a major independent factor associated with 3-year OS (HR 1.80; P = 0.008). Five-year OS was 25.6% (95%CI 14.9–37.6) for patients with complications and 45% (95%CI 33.3–53.4) for patients without.

Conclusions

Recent strategies facing advanced CLM include non-anatomic resections, portal-induced hypertrophy of the future remnant liver and aggressive medical preoperative treatments. CARe has the qualities of an approach that allows effective tumor clearance while maintaining good tolerance for the patient.