ArtiaX: An electron tomography toolbox for the interactive handling of sub‐tomograms in UCSF ChimeraX

Cryo‐electron tomography analysis involves the selection of macromolecular complexes to be used for subsequent sub‐tomogram averaging and structure determination. Here, we describe a plugin developed for UCSF ChimeraX that allows for the display, selection, and editing of particles within tomograms. Positions and orientations of selected particles can be manually set, modified and inspected in real time, both on screen and in virtual reality, and exported to various file formats. The plugin allows for the parallel visualization of particles stored in several meta data lists, in the context of any three‐dimensional image that can be opened with UCSF ChimeraX. The particles are rendered in user‐defined colors or using colormaps, such that individual classes or groups of particles, cross‐correlation coefficients, or other types of information can be highlighted to the user. The implemented functions are fast, reliable, and intuitive, exploring the broad range of features in UCSF ChimeraX. They allow for a fluent human–machine interaction, which enables an effective understanding of the sub‐tomogram processing pipeline, even for non‐specialist users.     

EGFR and HER2 exert distinct roles on colon cancer cell functional properties and expression of matrix macromolecules

Background

ErbB receptors, EGFR and HER2, have been implicated in the development and progression of colon cancer. Several intracellular pathways are mediated upon activation of EGFR and/or HER2 by EGF. However, there are limited data regarding the EGF-mediated signaling affecting functional cell properties and the expression of extracellular matrix macromolecules implicated in cancer progression.

Methods

Functional assays, such as cell proliferation, transwell invasion assay and migration were performed to evaluate the impact of EGFR/HER2 in constitutive and EGF-treated Caco-2 cells. Signaling pathways were evaluated using specific intracellular inhibitors. Western blot was also utilized to examine the phosphorylation levels of ERK1/2. Real time PCR was performed to evaluate gene expression of matrix macromolecules.

Results

EGF increases cell proliferation, invasion and migration and importantly, EGF mediates overexpression of EGFR and downregulation of HER2. The EGF–EGFR axis is the main pathway affecting colon cancer's invasive potential, proliferative and migratory ability. Intracellular pathways (PI3K-Akt, MEK1/2-Erk and JAK-STAT) are all implicated in the migratory profile. Notably, MT1- and MT2-MMP as well as TIMP-2 are downregulated, whereas uPA is upregulated via an EGF–EGFR network. The EGF–EGFR axis is also implicated in the expression of syndecan-4 and TIMP-1. However, glypican-1 upregulation by EGF is mainly mediated via HER2.

Conclusions and general significance

The obtained data highlight the crucial importance of EGF on the expression of both receptors and on the EGF–EGFR/HER2 signaling network, reveal the distinct roles of EGFR and HER2 on expression of matrix macromolecules and open a new area in designing novel agents in targeting colon cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Good‐bye to tropical alpine plant giants under warmer climates? Loss of range and genetic diversity in Lobelia rhynchopetalum

The main aim of this paper is to address consequences of climate warming on loss of habitat and genetic diversity in the enigmatic tropical alpine giant rosette plants using the Ethiopian endemic Lobelia rhynchopetalum as a model. We modeled the habitat suitability of L. rhynchopetalum and assessed how its range is affected under two climate models and four emission scenarios. We used three statistical algorithms calibrated to represent two different complexity levels of the response. We analyzed genetic diversity using amplified fragment length polymorphisms and assessed the impact of the projected range loss. Under all model and scenario combinations and consistent across algorithms and complexity levels, this afro‐alpine flagship species faces massive range reduction. Only 3.4% of its habitat seems to remain suitable on average by 2,080, resulting in loss of 82% (CI 75%–87%) of its genetic diversity. The remaining suitable habitat is projected to be fragmented among and reduced to four mountain peaks, further deteriorating the probability of long‐term sustainability of viable populations. Because of the similar morphological and physiological traits developed through convergent evolution by tropical alpine giant rosette plants in response to diurnal freeze‐thaw cycles, they most likely respond to climate change in a similar way as our study species. We conclude that specialized high‐alpine giant rosette plants, such as L. rhynchopetalum, are likely to face very high risk of extinction following climate warming.     

Proteoglycans in human laryngeal cartilage. Identification of proteoglycan types in successive cartilage extracts with particular reference to aggregating proteoglycans

The content, composition and structure of proteoglycans (PGs) in adult human laryngeal cartilage (HLC) were investigated. PGs were extracted from the tissue by using two different extraction protocols. In the first protocol, PGs were extracted under dissociative conditions, 4 M guanidine HCl (GdnHCl), and in the second protocol, sequentially, with phosphate buffered saline (PBS) and solutions of increasing GdnHCl concentration (0.5, 1, 2 and 4 M). Chemical and immunological analyses of dissociate extracts (first protocol) revealed the presence of four, at least, different types of PGs. Aggrecan was the major PG, versican, decorin and biglycan being in small amounts. Galactosaminoglycan-containing PGs (GalAGPGs) represented the vast majority of total PGs present in extracts of HLC. Differential digestion with chondroitinase ABC and AC II showed that the GalAGPGs from HLC contained a significant proportion of dermatan sulphate (DS). In addition, disaccharide analysis showed that 6-sulphated disaccharides predominated in chondroitin sulphate (CS) chains. The sequential extraction (second protocol) indicated that PBS extract contained very little amount of PGs. The 0.5, 1 and 2 M GdnHCl extracts contained 6.3%, 24.5% and 15.2% of total extracted PGs, respectively. Four molar GdnHCl extracted the larger proportion, about 53%, of total PGs. This extract contained almost only proteoglycan aggregate components i.e., G1 bearing aggrecan, hyaluronan and link protein. The characterization of the aggrecan showed that it constituted a polydisperse population of monomers with an average molecular mass of 720 kDa. The glycosaminoglycans (GAGs) present were chondroitin sulphate with a M(r) of 15 kDa, and keratan sulphate (KS) with a M(r) of 10 kDa, in proportions 84% and 16%, respectively.     

A meta-analysis of randomized controlled trials comparing endoscopic and open carpal tunnel decompression

Controversy exists regarding the benefit of endoscopic carpal tunnel release versus open carpal tunnel release in terms of grip/pinch strength, scar tenderness, pain, return to work, reversible/irreversible nerve damage, and adverse effects. Although a number of randomized controlled trials and systematic reviews have been published on the subject, to date, no large definitive randomized controlled trial or meta-analysis has been performed comparing endoscopic to open carpal tunnel release. This meta-analysis was undertaken to address the effectiveness of endoscopic carpal tunnel release relative to open carpal tunnel release. Key outcome measures from 13 randomized controlled trials were extracted and statistically combined. Heterogeneity was observed in three of the outcomes (i.e., grip strength, pain, and return to work), but the causes of heterogeneity could not be explained because of insufficient detail in the reported studies. Using the Jadad et al. scale, nine of 13 studies were of low methodologic quality. The effect sizes were compared between the studies that were rated as high quality and the studies that were rated as low quality on the Jadad et al. scale. Similarly, the studies that were rated as high quality on the Gerritsen et al. scale were compared with those that were rated as low quality. No clinically significant difference in effect sizes was apparent between studies of high and low methodologic quality. This meta-analysis supports the conclusion that endoscopic carpal tunnel release is favored over the open carpal tunnel release in terms of a reduction in scar tenderness and increase in grip and pinch strength at a 12-week follow-up. With regard to symptom relief and return to work, the data are inconclusive. Irreversible nerve damage is uncommon in either technique; however, there is an increased susceptibility to reversible nerve injury that is three times as likely to occur with endoscopic carpal tunnel release than with open carpal tunnel release.     

Segmentation of two- and three-dimensional data from electron microscopy using eigenvector analysis

An automatic image segmentation method is used to improve processing and visualization of data obtained by electron microscopy. Exploiting affinity criteria between pixels, e.g., proximity and gray level similarity, in conjunction with an eigenvector analysis, the image is subdivided into areas which correspond to objects or meaningful regions. Extending a proposal by Shi and Malik (1997, Proceedings of the IEEE conference on Computer Vision and Pattern Recognition, pp. 731-737) the approach was adapted to the field of electron microscopy, especially to three-dimensional application as needed by electron tomography. Theory, implementation, parameter setting, and results obtained with a variety of data are presented and discussed. The method turns out to be a powerful tool for visualization with the potential for further improvement by developing and tuning new affinity.     

Inhibition of microsurgical thrombosis by the platelet glycoprotein IIb/IIIa antagonist SR121566A

Despite major improvements in tools and significant refinements of techniques, microsurgical anastomosis still carries a significant risk of failure due to microvascular thrombosis. The key to improving the success of microvascular surgery may lie in the pharmacologic control of thrombus formation. Central to pathologic arterial thrombosis are platelets. Glycoprotein IIb/IIIa is a highly abundant platelet surface receptor that plays a major role in platelet aggregation by binding platelets to each other through the coagulation factor fibrinogen. To explore the ability of antithrombotic agents to prevent microvascular thrombosis, a rabbit ear artery model was used in which a standardized arterial injury results in predictable thrombus formation. This model was used to examine whether SR121566A, a specific and potent glycoprotein IIb/IIIa inhibitor, can successfully prevent microsurgical thrombosis.Using a coded, double-blind experimental design, 20 rabbits (40 arteries) were assigned to four treatment groups: (1) saline injection (n = 10), (2) acetylsalicylic acid 10 mg/kg (n = 10), (3) heparin 0.5 mg/kg bolus with subsequent intermittent boluses of 0.25 mg/kg every 30 minutes (n = 10), and (4) SR121566A 2 mg/kg bolus (n = 10). After vessel damage and clamp release, arteries were assessed for patency at 5, 30, and 120 minutes by the Acland refill test. Coagulation assays, in vivo bleeding times, and ex vivo platelet aggregation studies were also conducted. Scanning electron microscopy was used to examine mural thrombus composition.A significant, fourfold increase in vessel patency following administration of SR121566A over saline control (80 percent versus 20 percent patency, respectively, at 35 minutes after reperfusion, p < 0.01) was noted. This was correlated with marked inhibition of ex vivo platelet aggregation. This antiplatelet treatment did not prolong coagulation assays (mean international normalized ratio: saline, 0.66 +/- 0.04; SR121566A, 0.64 +/- 0.03; mean thromboplastin time: saline, 19.63 +/- 0.67; SR121566A, 17.87 +/- 3.27) and bleeding times (mean bleeding time: saline, 42 +/- 4; SR121566A, 48 +/- 6). Scanning electron microscopy demonstrated extensive platelet and fibrin deposition in control vessel thrombi. In contrast, thrombi from SR121566A-treated vessels demonstrated predominance of fibrin with few platelets when examined under scanning electron microscopy.Administration of SR121566A was associated with a significant increase in vessel patency, without deleterious effects on coagulation assays or bleeding times. The increase in vessel patency was correlated with inhibition of platelet aggregation and decreased platelet deposition, as demonstrated by scanning electron microscopy. Glycoprotein IIb/IIIa antagonists represent a new class of anti-platelet agents that may be suited for inhibiting microsurgical thrombosis. This study supports further investigation into the use of these agents in microsurgery.     

Study of changes in physicochemical and microbiological characteristics of shrimps (Melicertus kerathurus) stored under modified atmosphere packaging

Fresh minimally processed shrimps were stored under modified atmosphere packaging (60% CO₂:40% N₂ for MAP A and 92.9% N₂:5.1% CO₂:2% O₂ for MAP B) for 5 days at 3 °C. Total mesophiles, H₂S forming bacteria, Pseudomonas spp., Brochothrix thermosphacta, firmness, color and sensory parameters were investigated throughout the whole time of the experiment. During storage period samples stored under MAP B managed to retain firmness values close to the initial values. All microbial populations growth was suppressed by the presence of MAP A. Samples stored under MAP B managed to maintain their firmness values close to the initial ones while MAP A samples were significantly less firm (p < 0.05).     

Differentiation of myomas by means of biomagnetic and doppler findings

Aim

To elucidate the hemodynamics of the uterine artery myomas by use of Doppler ultrasound and biomagnetic measurements.

Method

Twenty-four women were included in the study. Sixteen of them were characterised with large myomas whereas 8 of them with small ones. Biomagnetic signals of uterine arteries myomas were recorded and analyzed with Fourier analysis. The biomagnetic signals were distributed according to spectral amplitudes as high (140–300 ft/√Hz), low (50–110 ft/√Hz) and borderline (111–139 ft/√Hz). Uterine artery waveform measurements were evaluated by use of Pulsatility Index (PI) (normal value PI < 1.45).

Results

There was a statistically significant difference between large and small myomas concerning the waveform amplitudes (P < 0.0005) and the PI index (P < 0.0005). Specifically, we noticed high biomagnetic amplitudes in most large myomas (93.75 %) and low biomagnetic amplitudes in most small ones (87.5 %).

Conclusion

It is suggested that the biomagnetic recordings of uterine artery myomas could be a valuable modality in the estimation of the circulation of blood cells justifying the findings of Doppler velocimetry examination.