A perspective of fluorescence microscopy for cellular structural biology with EGFR as witness.

The epidermal growth factor receptor (EGFR) is a poster child for the understanding of receptor behaviour, and of paramount importance to cell function and human health. Cloned almost forty years ago, the interest in EGFR's structure/function relationships remains unabated, not least because changes in oncogenic EGFR mutants are key drivers of the formation of lung and brain tumours. The structure of the assemblies formed by EGFR have been comprehensibly investigated by techniques such as high-resolution X-ray crystallography, NMR and all-atom molecular dynamics (MD) simulations. However, the complexity embedded in the portfolio of EGFR states that are only possible in the physiological environment of cells has often proved refractory to cell-free structural methods. Conversely, some key inroads made by quantitative fluorescence microscopy and super-resolution have depended on exploiting the wealth of structures available. Here, a brief personal perspective is provided on how quantitative fluorescence microscopy and super-resolution methods have cross-fertilised with cell-free-derived EGFR structural information. I primarily discuss areas in which my research group has made a contribution to fill gaps in EGFR's cellular structural biology and towards developing new tools to investigate macromolecular assemblies in cells.

A brief history of the octopus imaging facility to celebrate its 10th anniversary.

Octopus (Optics Clustered to OutPut Unique Solutions) celebrated in June 2020 its 10th birthday. Based at Harwell, near Oxford, Octopus is an open access, peer reviewed, national imaging facility that offers successful U.K. applicants supported access to single molecule imaging, confocal microscopy, several flavours of superresolution imaging, light sheet microscopy, optical trapping and cryoscanning electron microscopy. Managed by a multidisciplinary team, Octopus has so far assisted >100 groups of U.K. and international researchers. Cross-fertilisation across fields proved to be a strong propeller of success underpinned by combining access to top-end instrumentation with a strong programme of imaging hardware and software developments. How Octopus was born, and highlights of the multidisciplinary output produced during its 10-year journey are reviewed below, with the aim of celebrating a myriad of collaborations with the U.K. scientific community, and reflecting on their scientific and societal impact.

Endothelial dysfunction is an early indicator of sepsis and neutrophil degranulation of septic shock in surgical patients.

BACKGROUND: Stratification of the severity of infection is currently based on the Sequential Organ Failure Assessment (SOFA) score, which is difficult to calculate outside the ICU. Biomarkers could help to stratify the severity of infection in surgical patients. METHODS: Levels of ten biomarkers indicating endothelial dysfunction, 22 indicating emergency granulopoiesis, and six denoting neutrophil degranulation were compared in three groups of patients in the first 12 h after diagnosis at three Spanish hospitals. RESULTS: There were 100 patients with infection, 95 with sepsis and 57 with septic shock. Seven biomarkers indicating endothelial dysfunction (mid-regional proadrenomedullin (MR-ProADM), syndecan 1, thrombomodulin, angiopoietin 2, endothelial cell-specific molecule 1, vascular cell adhesion molecule 1 and E-selectin) had stronger associations with sepsis than infection alone. MR-ProADM had the highest odds ratio (OR) in multivariable analysis (OR 11·53, 95 per cent c.i. 4·15 to 32·08; P = 0·006) and the best area under the curve (AUC) for detecting sepsis (0·86, 95 per cent c.i. 0·80 to 0·91; P < 0·001). In a comparison of sepsis with septic shock, two biomarkers of neutrophil degranulation, proteinase 3 (OR 8·09, 1·34 to 48·91; P = 0·028) and lipocalin 2 (OR 6·62, 2·47 to 17·77; P = 0·002), had the strongest association with septic shock, but lipocalin 2 exhibited the highest AUC (0·81, 0·73 to 0·90; P < 0·001). CONCLUSION: MR-ProADM and lipocalin 2 could be alternatives to the SOFA score in the detection of sepsis and septic shock respectively in surgical patients with infection.

ANTECEDENTES: La estratificación de la gravedad de una infección se basa actualmente en la puntuación SOFA (Sequential Organ Failure Assessment), que es difícil de calcular fuera de la unidad de cuidados intensivos. Los biomarcadores podrían ayudar a estratificar la gravedad de la infección en pacientes quirúrgicos. MÉTODOS: Se compararon las concentraciones de 10 biomarcadores que denotan disfunción endotelial, 22 que indican granulopoyesis de emergencia y 6 que expresan la degranulación de neutrófilos en tres grupos de pacientes de tres hospitales españoles (100 con infección, 95 con sepsis y 57 con shock séptico) en las primeras doce horas después del diagnóstico. RESULTADOS: Siete biomarcadores que expresan disfunción endotelial (proadrenomedulina, sindecan-1, trombomodulina, angiopoyetina-2, endocan-1, molécula de adhesión endotelial 1 y E-selectina) mostraron una fuerte asociación con la sepsis en comparación con la infección aislada. La proadrenomedulina presentó el valor más alto de la razón de oportunidades (odds ratio, OR) en el análisis multivariable (OR 11,53, i.c. del 95% 4,15-32,08, P = 0,006) y la mejor área bajo la curva para detectar sepsis (AUC 0,86, i.c. del 95% 0,80-0,91, P < 0,001). En la comparación entre sepsis y shock séptico, los biomarcadores que mostraron la asociación más estrecha con el shock séptico fueron dos biomarcadores de degranulación de neutrófilos (proteinasa-3 y lipocalina-2) (OR 8,09, i.c. del 9% 1,34-48,91, P = 0,028; OR 6.62, i.c. del 95% 2,47-17,77, P = 0,002), pero la lipocalina-2 presentó la mejor AUC (0,81, i.c. del 95% 0,73-0,90, P < 0,001). CONCLUSIÓN: la proadrenomedulina y la lipocalina-2 podrían representar alternativas a la puntuación SOFA para detectar sepsis y shock séptico en pacientes quirúrgicos con infección.

The cell wall regulates dynamics and size of plasma-membrane nanodomains in Arabidopsis.

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signaling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using subdiffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains.

Effects of Cyclosporine, Tacrolimus, and Rapamycin on Osteoblasts.

PURPOSE: One factor that can contribute to severe bone loss after transplantation is the direct action of immunosuppressants on bone cells. The aim of this work was to study the effects of cyclosporine (CsA), tacrolimus (FK-506), and rapamycin (RAPA) on the release of three local factors directly implicated in bone-remodeling regulation and apoptosis of human osteoblasts: interleukin (IL)-6, osteoprotegerin, and receptor activator of nuclear factor κß (RANKL). BASIC PROCEDURES: Human osteoblasts were obtained from five different patients who underwent orthopedic surgery. These cells were treated with what are considered to be a clinically high dose and an acceptable dose of each immunosuppressant-RAPA 50 ng/mL and 12 ng/mL, FK-506 20 ng/mL and 5 ng/mL, CsA 1000 ng/mL and 250 ng/mL-or vehicle. Apoptotic cell death was quantified using flow cytometry of DNA content in permeabilized, propidium iodide-stained cells. IL-6 was measured using enzyme-linked immunosorbent assay (ELISA; Quantikine Human IL6, R&D Systems, Minneapolis, Minn, United States). Messenger RNA (mRNA) expression of osteoprotegerin, RANKL, and IL-6 was measured using quantitative RT-PCR. MAIN FINDINGS: A significant increase in IL-6 (mRNA and released protein) was observed in the presence of FK-506 and RAPA. Addition of RAPA to the cultures of osteoblasts produced a significant increase in the OPG/RANKL ratio. A significant increase in osteoblast apoptosis was observed in the cells treated with FK-506 and RAPA 24 hours after the addition of immunosuppressants. CsA did not produce any significant changes in osteoblasts. PRINCIPAL CONCLUSIONS: These results suggest that an increase in osteoblast apoptosis by osteoblasts may be one of the mechanisms by which bone loss occurs after RAPA and FK-506 treatments.

Potential risks of using cement-augmented screws for spinal fusion in patients with low bone quality.

BACKGROUND CONTEXT: Dramatic increases in the average life expectancy have led to increases in the variety of degenerative changes and deformities observed in the aging spine. The elderly population can present challenges for spine surgeons, not only because of increased comorbidities, but also because of the quality of their bones. Pedicle screws are the implants used most commonly in spinal surgery for fixation, but their efficacy depends directly on bone quality. Although polymethyl methacrylate (PMMA)-augmented screws represent an alternative for patients with osteoporotic vertebrae, their use has raised some concerns because of the possible association between cement leakages (CLs) and other morbidities. PURPOSE: To analyze potential complications related to the use of cement-augmented screws for spinal fusion and to investigate the effectiveness of using these screws in the treatment of patients with low bone quality. STUDY DESIGN: A retrospective single-center study. PATIENT SAMPLE: This study included 313 consecutive patients who underwent spinal fusion using a total of 1,780 cement-augmented screws. METHODS AND OUTCOME MEASURES: We analyzed potential complications related to the use of cement-augmented screws, including CL, vascular injury, infection, screw extraction problems, revision surgery, and instrument failure. There are no financial conflicts of interest to report. RESULTS: A total of 1,043 vertebrae were instrumented. Cement leakage was observed in 650 vertebrae (62.3%). There were no major clinical complications related to CL, but two patients (0.6%) had radicular pain related to CL at the S1 foramina. Of the 13 patients (4.1%) who developed deep infections requiring surgical debridement, two with chronic infections had possible spondylitis that required instrument removal. All patients responded well to antibiotic therapy. Revision surgery was performed in 56 patients (17.9%), most of whom had long construction. A total of 180 screws were removed as a result of revision. There were no problems with screw extraction. CONCLUSIONS: These results demonstrate the efficacy and safety of cement-augmented screws for the treatment of patients with low bone mineral density.

Effect of early conversion to everolimus together with prophylaxis with valganciclovir in the prevention of cytomegalovirus infection in heart transplant recipients.

INTRODUCTION: Viral infections, especially cytomegalovirus (CMV), are a leading cause of early death and morbidity after heart transplantation. Several strategies have been used to minimize the risk, including universal prophylaxis with ganciclovir or valganciclovir and preemptive therapy. Lately, everolimus (EVE) efficacy studies have shown a protective effect against CMV infection. METHODS: We studied retrospectively a series of 223 heart transplant patients, dividing them into 5 groups according to CMV prevention strategy: 16 patients were at low risk for infection (negative recipient [R-]/negative donor [D-]) and received no treatment; 26 patients received prophylactic therapy with ganciclovir, 8 patients prophylaxis with valganciclovir, 145 patients received preemptive therapy and 28 patients prophylaxis with valganciclovir and early conversion to EVE. RESULTS: There were no cases of CMV infection in the low-risk group. There was 1 case of CMV infection in the group that received valganciclovir and conversion to EVE. Among the patients who received prophylaxis with ganciclovir or valganciclovir or preemptive therapy, CMV infection was detected in 68 patients (37%). CONCLUSIONS: Early conversion to EVE in addition to valganciclovir prophylaxis was superior to other strategies in our series for the prevention of CMV infection.

A 'pocket guide' to total internal reflection fluorescence.

The phenomenon of total internal reflection fluorescence (TIRF) was placed in the context of optical microscopy by Daniel Axelrod over three decades ago. TIRF microscopy exploits the properties of an evanescent electromagnetic field to optically section sample regions in the close vicinity of the substrate where the field is induced. The first applications in cell biology targeted investigation of phenomena at the basolateral plasma membrane. The most notable application of TIRF is single-molecule experiments, which can provide information on fluctuation distributions and rare events, yielding novel insights on the mechanisms governing the molecular interactions that underpin many fundamental processes within the cell. This short review intends to provide a 'one stop shop' explanation of the electromagnetic theory behind the remarkable properties of the evanescent field, guide the reader through the principles behind building or choosing your own TIRF system and consider how the most popular applications of the method exploit the evanescent field properties.

Usefulness of bone turnover markers as predictors of mortality risk, disease progression and skeletal-related events appearance in patients with prostate cancer with bone metastases following treatment with zoledronic acid: TUGAMO study.

BACKGROUND: Owing to the limited validity of clinical data on the treatment of prostate cancer (PCa) and bone metastases, biochemical markers are a promising tool for predicting survival, disease progression and skeletal-related events (SREs) in these patients. The aim of this study was to evaluate the predictive capacity of biochemical markers of bone turnover for mortality risk, disease progression and SREs in patients with PCa and bone metastases undergoing treatment with zoledronic acid (ZA). METHODS: This was an observational, prospective and multicenter study in which ninety-eight patients were included. Patients were treated with ZA (4 mg every 4 weeks for 18 months). Data were collected at baseline and 3, 6, 9, 12, 15 and 18 months after the beginning of treatment. Serum levels of bone alkaline phosphtase (BALP), aminoterminal propeptide of procollagen type I (P1NP) and beta-isomer of carboxiterminal telopeptide of collagen I (ß-CTX) were analysed at all points in the study. Data on disease progression, SREs development and survival were recorded. RESULTS: Cox regression models with clinical data and bone markers showed that the levels of the three markers studied were predictive of survival time, with ß-CTX being especially powerful, in which a lack of normalisation in visit 1 (3 months after the beginning of treatment) showed a 6.3-times more risk for death than in normalised patients. Levels of these markers were also predictive for SREs, although in this case BALP and P1NP proved to be better predictors. We did not find any relationship between bone markers and disease progression. CONCLUSION: In patients with PCa and bone metastases treated with ZA, ß-CTX and P1NP can be considered suitable predictors for mortality risk, while BALP and P1NP are appropriate for SREs. The levels of these biomarkers 3 months after the beginning of treatment are especially important.

Unsupervised 4D myocardium segmentation with a Markov Random Field based deformable model.

A stochastic deformable model is proposed for the segmentation of the myocardium in Magnetic Resonance Imaging. The segmentation is posed as a probabilistic optimization problem in which the optimal time-dependent surface is obtained for the myocardium of the heart in a discrete space of locations built upon simple geometric assumptions. For this purpose, first, the left ventricle is detected by a set of image analysis tools gathered from the literature. Then, the segmentation solution is obtained by the Maximization of the Posterior Marginals for the myocardium location in a Markov Random Field framework which optimally integrates temporal-spatial smoothness with intensity and gradient related features in an unsupervised way by the Maximum Likelihood estimation of the parameters of the field. This scheme provides a flexible and robust segmentation method which has been able to generate results comparable to manually segmented images for some derived cardiac function parameters in a set of 43 patients affected in different degrees by an Acute Myocardial Infarction.

Probabilistic-driven oriented Speckle reducing anisotropic diffusion with application to cardiac ultrasonic images.

A novel anisotropic diffusion filter is proposed in this work with application to cardiac ultrasonic images. It includes probabilistic models which describe the probability density function (PDF) of tissues and adapts the diffusion tensor to the image iteratively. For this purpose, a preliminary study is performed in order to select the probability models that best fit the stastitical behavior of each tissue class in cardiac ultrasonic images. Then, the parameters of the diffusion tensor are defined taking into account the statistical properties of the image at each voxel. When the structure tensor of the probability of belonging to each tissue is included in the diffusion tensor definition, a better boundaries estimates can be obtained instead of calculating directly the boundaries from the image. This is the main contribution of this work. Additionally, the proposed method follows the statistical properties of the image in each iteration. This is considered as a second contribution since state-of-the-art methods suppose that noise or statistical properties of the image do not change during the filter process.

Sequential anisotropic multichannel Wiener filtering with Rician bias correction applied to 3D regularization of DWI data.

It has been shown that the tensor calculation is very sensitive to the presence of noise in the acquired images, yielding to very low quality Diffusion Tensor Images (DTI) data. Recent investigations have shown that the noise present in the Diffusion Weighted Images (DWI) causes bias effects on the DTI data which cannot be corrected if the noise characteristic is not taken into account. One possible solution is to increase the minimum number of acquired measurements (which is 7) to several tens (or even several hundreds). This has the disadvantage of increasing the acquisition time by one (or two) orders of magnitude, making the process inconvenient for a clinical setting. We here proposed a turn-around procedure for which the number of acquisitions is maintained but, the DWI data are filtered prior to determining the DTI. We show a significant reduction on the DTI bias by means of a simple and fast procedure which is based on linear filtering; well-known drawbacks of such filters are circumvented by means of anisotropic neighborhoods and sequential application of the filter itself. Information of the first order probability density function of the raw data, namely, the Rice distribution, is also included. Results are shown both for synthetic and real datasets. Some error measurements are determined in the synthetic experiments, showing how the proposed scheme is able to reduce them. It is worth noting a 50% increase in the linear component for real DTI data, meaning that the bias in the DTI is considerably reduced. A novel fiber smoothness measure is defined to evaluate the resulting tractography for real DWI data. Our findings show that after filtering, fibers are considerably smoother on the average. Execution times are very low as compared to other reported approaches which allows for a real-time implementation.

Simultaneous widefield single molecule orientation and FRET microscopy in cells.

We combine single molecule fluorescence orientation imaging with single-pair fluorescence resonance energy transfer microscopy, using a total internal reflection microscope. We show how angles and FRET efficiencies can be determined for membrane proteins at the single molecule level and provide data from the epidermal growth factor receptor system in cells.

[Peritoneal dialyisis role in heart failure treatment, experience in our center]. / Papel de la diálisis peritoneal en el tratamiento de la insuficiencia cardíaca. Experiencia en nuestro centro.

Peritoneal dialysis is a renal replacement therapy indicated in patients with an unstable hemodynamic status. It has been used, by ultrafiltration, preferably in those patients with congestive heart failure refractory to conventional medical therapy. We present the experience of our center with five patients who were affected by severe congestive heart failure [Class IV on the New York Heart Association (NYHA) scale] and diverse stages of chronic renal failure, who received this therapy. Icodextrin has been used as an osmotic agent to induce ultrafiltration. The follow-up period ranged between 5 and 14 months (9.8 +/- 3.7 months). The results that we have found are similar to those of other studies: we observed a significant improvement in quality of life and a reduction in morbidity and hospitalization rates in all our patients. But it seems to be necessary to make a prospective randomized controlled trial with more number of individuals to confirm these promising facts, to clarify the impact on the survival, and to analyze the cost-benefit for treating patients suffering from refractory, end stage congestive heart failure.

Multidimensional single-molecule imaging in live cells using total-internal-reflection fluorescence microscopy.

We have developed a wide-field total-internal-reflection fluorescence microscope capable of imaging single molecules in live cells, resolved in both wavelength and polarization. We show fluorescence resonance energy transfer between single pairs of fluorescent molecules bound to signaling receptors in the plasma membrane of live cells and demonstrate the importance of polarization discrimination in addition to wavelength separation.

Real-time studies of the interactions between epidermal growth factor and its receptor during endocytic trafficking.

The interactions of growth factors with cell surface receptors regulate fundamental cell processes, such as growth, differentiation and transformation. Understanding the nature of these interactions at the molecular level is of fundamental importance in cell biology. This is not only from the point of view of basic science, but also because of the repercussions such knowledge might have in understanding the mode of action of drugs in cells. Receptor mediated endocytosis has been implicated in the downregulation of the mitogenic signal. However, no data are thus far available on how growth factor/receptor interactions might control endocytic trafficking. Here we show that information on modes of binding and receptor conformational changes can be obtained using time-resolved fluorescence methods. We have found that fluorescent probes bound to epidermal growth factor (EGF) show dynamic fluorescence quenching when EGF is bound to internalising EGF receptors (EGFR). We propose that this dynamic quenching takes place because EGF-bound probes interact with tryptophan residues in the extracellular domain of the EGF-EGFR complex. Real-time accumulation of fluorescent decays has also allowed us to follow the time course of a conformational change in EGFR occurring during endocytosis, and correlate this information with endosomal trafficking and EGFR recycling.

[Renal cell carcinoma. Clinical onset with peritoneal carcinomatosis. Report of a case]. / Carcinoma de células renales. Presentación clínica debut con carcinomatosis peritoneal. A propósito de un caso.

Renal carcinoma accounts for 2-3% of malignant tumours in the adult, with high tendency for metastasis basically in liver, lymph nodes, lungs and bones. Intraperitoneal, gut, mesentery and omentum involvement is extremely rare affecting only 1% of patients with metastasis at post-mortem. Contribution of one case of renal carcinoma with initial presentation as intraperitoneal metastasis.

Time-resolved X-ray diffraction studies of myosin head movements in live frog sartorius muscle during isometric and isotonic contractions.

Using the facilities at the Daresbury Synchrotron Radiation Source, meridional diffraction patterns of muscles at ca 8 degrees C were recorded with a time resolution of 2 or 4 ms. In isometric contractions tetanic peak tension (P0) is reached in ca 400 ms. Under such conditions, following stimulation from rest, the timing of changes in the major reflections (the 38.2 nm troponin reflection, and the 21.5 and 14.34/14.58 nm myosin reflections) can be explained in terms of four types of time courses: K1, K2, K3 and K4. The onset of K1 occurs immediately after stimulation, but that of K2, K3 and K4 is delayed by a latent period of ca 16 ms. Relative to the end of their own latent periods the half-times for K1, K2, K3 and K4 are 14-16, 16, 32 and 52 ms, respectively. In half-times, K1, K2, K3 lead tension rise by 52, 36 and 20 ms, respectively. K4 parallels the time course of tension rise. From an analysis of the data we conclude that K1 reflects thin filament activation which involves the troponin system; K2 arises from an order-disorder transition during which the register between the filaments is lost; K3 is due to the formation of an acto-myosin complex which (at P0) causes 70% or more of the heads to diffract with actin-based periodicities; and K4 is caused by a change in the axial orientation of the myosin heads (relative to thin filament axis) which is estimated to be from 65-70 degrees at rest to ca 90 degrees at P0. Isotonic contraction experiments showed that during shortening under a load of ca 0.27 P0, at least 85% of the heads (relative to those forming an acto-myosin complex at P0) diffract with actin-based periodicities, whilst their axial orientation does not change from that at rest. During shortening under a negligible load, at most 5-10% of the heads (relative to those forming an acto-myosin complex at P0) diffract with actin-based periodicities, and their axial orientation also remains the same as that at rest. This suggests that in isometric contractions the change in axial orientation is not the cause of active tension production, but rather the result of it. Analysis of the data reveals that independent of load, the extent of asynchronous axial motions executed by most of the cycling heads is no more than 0.5-0.65 nm greater than at rest.(ABSTRACT TRUNCATED AT 400 WORDS)