Bridging structural and cell biology with cryo-electron microscopy.

Most life scientists would agree that understanding how cellular processes work requires structural knowledge about the macromolecules involved. For example, deciphering the double-helical nature of DNA revealed essential aspects of how genetic information is stored, copied and repaired. Yet, being reductionist in nature, structural biology requires the purification of large amounts of macromolecules, often trimmed off larger functional units. The advent of cryogenic electron microscopy (cryo-EM) greatly facilitated the study of large, functional complexes and generally of samples that are hard to express, purify and/or crystallize. Nevertheless, cryo-EM still requires purification and thus visualization outside of the natural context in which macromolecules operate and coexist. Conversely, cell biologists have been imaging cells using a number of fast-evolving techniques that keep expanding their spatial and temporal reach, but always far from the resolution at which chemistry can be understood. Thus, structural and cell biology provide complementary, yet unconnected visions of the inner workings of cells. Here we discuss how the interplay between cryo-EM and cryo-electron tomography, as a connecting bridge to visualize macromolecules in situ, holds great promise to create comprehensive structural depictions of macromolecules as they interact in complex mixtures or, ultimately, inside the cell itself.

La contaminación citológica de la aguja de punción en ecobroncoscopia / Cytologic Contamination of the Sampling Needle in Endobronchial Ultrasound

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[Translated article] Cytologic Contamination of the Sampling Needle in Endobronchial Ultrasound / La contaminación citológica de la aguja de punción en ecobroncoscopia

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Resultado de los medios diagnósticos en pacientes operados de cáncer de tiroides. Hospital Universitario Comandante "Faustino Pérez Hernández". Matanzas / Outcomes of the diagnostic means in patients undergoing thyroid cancer surgery. University Hospital "Comandante Faustino Pérez Hernández". Matanzas

RESUMEN Introducción: el cáncer de tiroides representa un 1% del total de todos los tipos de cáncer. Su incidencia parece aumentar un 4% cada año, y en la actualidad es el octavo cáncer más frecuente en mujeres. Se utilizan medios diagnósticos para definir que paciente operar y la estrategia terapéutica a seguir. Objetivo: evaluar el resultado de los medios diagnósticos en los pacientes operados de cáncer de tiroides. Materiales y métodos: se realizó un estudio descriptivo, retrospectivo y observacional que incluye todos los pacientes operados de cáncer tiroides en el servicio de Cirugía General del Hospital Universitario "Comandante Faustino Pérez Hernández", en el período desde enero de 1993 a diciembre del 2018. Se confeccionó una planilla pararecopilar la información para el estudio de la base de datos y los resultados se presentaron en tablas de frecuencia, números y porciento. Resultados: el cáncer de tiroides fue más frecuente en el grupo etario de 31 a 50 años, predomino el sexo femenino, la variedad histológica papilar fue la más frecuente, el ultrasonido y la citología con aguja fina aportan resultados favorables para definir la estirpe biológica de los tumores del tiroides, no comportándose de igual forma la biopsia por congelación. Conclusiones: el cáncer de tiroides es más frecuente en pacientes relativamente jóvenes, del sexo femenino y de variedad papilar. El ultrasonido y la citología con aguja fina aportan criterios beneficiosos para definir conducta terapéutica (AU).

SUMMARY Introduction: thyroid cancer represents 1% of the total of all kinds of cancer. Its incidence seems to increase 4% every year, and at the present time it is the eighth more frequent cancer in women. Diagnostic means are used to determine what patient undergoes a surgery and the therapeutic strategy to follow. Objective: to evaluate the results of the diagnostic means used in patients who undergo a thyroid cancer surgery. Materials and methods: a retrospective, descriptive, observational study was carried out including all patients who underwent a surgery of thyroid cancer in the service of General Surgery of the University Hospital ¨Comandante Faustino Perez Hernandez¨ in the period from January 1993 to December 2018. A form was elaborated to collect the information for the study of the database; the results were presented in charts of frequency, numbers and percentages. Results: the thyroid cancer was more frequent in the age group of 31 to 50 years; the female sex prevailed; the most frequent variety was the histological papillary one; ultrasound and fine needle cytology yield favourable results to define the biological stock of the thyroid cancers unlike the behaviour of the biopsy by freezing. Conclusions: thyroid cancer of the papillary variety is more frequent in relatively young, female patients. Ultrasound and fine needle cytology yield beneficial criteria to define the therapeutic behaviour (AU).

Clinical-Cytological-Grading and phenotyping in patients with chronic rhinosinusitis with nasal polyps: the relevance in clinical practice.

Chronic rhinosinusitis (CRS) includes two main phenotypes: without nasal polyps (CRSsNP) and with nasal polyps (CRSwNP). CRSwNP may be associated with comorbidity, mainly concerning asthma, aspirin intolerance, and allergy. CRSwNP patients may also be evaluated by clinical-cytological grading (CCG). The current study investigated the prevalence and characteristics of the different CCG and phenotypes in CRSwNP outpatients examined in clinical practice. This retrospective cross-sectional study enrolled 791 consecutive CRSwNP outpatients (424 males, mean age 48.8 years). In the total population, asthma was a common comorbidity (30.8%) as well as aspirin intolerance (24.8%), and allergy (50.8%). As concerns CCG-grading, 210 (26.5%) outpatients had low-grade, 366 (46.3%) medium, and 215 (27.2%) high. As regards cytological phenotypes, 87 (11%) had neutrophilic type, 371 (46.3%) eosinophilic, 112 (14.2%) mast cell, and 221 (27.9%) mixed. High-grade CCG was significantly associated with more frequent asthma, aspirin intolerance, allergy, recurrent surgery, and mixed cytological phenotype. Low-grade CCG was characterized by fewer comorbidities and operations, and neutrophilic phenotype. Therefore, the present study confirmed that CCG is a useful tool in the management of outpatients with CRSwNP. CRSwNP is frequently associated with asthma, aspirin intolerance, and allergy comorbidity. High-grade CCG is frequently characterized by a mixed cytological phenotype, thus, by more severe progress. These real-world outcomes underline that CRSwNP deserves adequate attention for careful management and optimal identification of the best-tailored therapy; CCG and cytological phenotyping could be fruitful tools in clinical practice. Asthma and aspirin intolerance should be adequately investigated in all CRS patients.

A homogeneous bioluminescent immunoassay to probe cellular signaling pathway regulation.

Monitoring cellular signaling events can help better understand cell behavior in health and disease. Traditional immunoassays to study proteins involved in signaling can be tedious, require multiple steps, and are not easily adaptable to high throughput screening (HTS). Here, we describe a new immunoassay approach based on bioluminescent enzyme complementation. This immunoassay takes less than two hours to complete in a homogeneous "Add and Read" format and was successfully used to monitor multiple signaling pathways' activation through specific nodes of phosphorylation (e.g pIκBα, pAKT, and pSTAT3). We also tested deactivation of these pathways with small and large molecule inhibitors and obtained the expected pharmacology. This approach does not require cell engineering. Therefore, the phosphorylation of an endogenous substrate is detected in any cell type. Our results demonstrate that this technology can be broadly adapted to streamline the analysis of signaling pathways of interest or the identification of pathway specific inhibitors.

Super-resolution microscopy demystified.

Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.

Microfluidic chambers using fluid walls for cell biology.

Many proofs of concept have demonstrated the potential of microfluidics in cell biology. However, the technology remains inaccessible to many biologists, as it often requires complex manufacturing facilities (such as soft lithography) and uses materials foreign to cell biology (such as polydimethylsiloxane). Here, we present a method for creating microfluidic environments by simply reshaping fluids on a substrate. For applications in cell biology, we use cell media on a virgin Petri dish overlaid with an immiscible fluorocarbon. A hydrophobic/fluorophilic stylus then reshapes the media into any pattern by creating liquid walls of fluorocarbon. Microfluidic arrangements suitable for cell culture are made in minutes using materials familiar to biologists. The versatility of the method is demonstrated by creating analogs of a common platform in cell biology, the microtiter plate. Using this vehicle, we demonstrate many manipulations required for cell culture and downstream analysis, including feeding, replating, cloning, cryopreservation, lysis plus RT-PCR, transfection plus genome editing, and fixation plus immunolabeling (when fluid walls are reconfigured during use). We also show that mammalian cells grow and respond to stimuli normally, and worm eggs develop into adults. This simple approach provides biologists with an entrée into microfluidics.

Concomitant use of polarization and negative phase contrast microscopy for the study of microorganisms.

A simultaneous application of negative phase contrast and polarization microscopy was used to study the internal structure of microbial cells. Negative phase contrast allowed us to display the fine cell structures with a refractive index of light approaching that of the environment, e.g., the cytoplasm, and converted an invisible phase image to a visible amplitude one. In the polarizing microscope, cross-polarizing filters, together with first-order quartz compensator and a turntable, showed maximum birefringence of individual structures. Material containing algae was collected in ponds in the villages Sýkorice and Zbecno (Protected Landscape Area Krivoklátsko). Objects were studied in a laboratory microscope (Carl Zeiss Jena, type NfpK), equipped with a basic body In Ph 160 with an exchangeable module Ph, LOMO St. Petersburg turntable mounted on a centering holder of our own construction and a Nikon D 70 digital SLR camera. Anisotropic granules were found only in the members of two orders of algae (Euglenales, Euglenophyceae and Chlorococcales, Chlorophyceae). They always showed strong birefringence and differed in both number and size. An important finding concerned thin pellicles in genus Euglena (Euglenales, Euglenophyceae) which exhibited weak birefringence. In genus Pediastrum (Chlorococcales, Chlorophyceae), these granules were found only in living coenobium cells. In contrast, dead coenobium cells contained many granules without birefringence-an important finding. Another important finding included birefringent lamellar structure of the transverse cell wall and weak birefringence of pyrenoids in filamentous algae of genus Spirogyra (Zygnematales, Conjugatophyceae). It was clearly displayed by the negative phase contrast and has not been documented by other methods. This method can also record the very weak birefringence of the frustule of a diatom of genus Pinnularia (Naviculales, Bacillariophyceae), which was further reinforced by the use of quartz compensator-an important finding. Simultaneous use of negative phase contrast and polarization microscopy allowed us to study not only birefringent granules of storage substances in microorganisms, but also the individual lamellae of the cell walls of filamentous algae and very thin frustule walls in diatoms. These can be visualized only by this contrast method, which provides a higher resolution (subjective opinion only) than other methods such as positive phase contrast or relief contrast.

DIY: "Do Imaging Yourself" - Conventional microscopes as powerful tools for in vivo investigation.

Intravital imaging has been increasingly employed in cell biology studies and it is becoming one of the most powerful tools for in vivo investigation. Although some protocols can be extremely complex, most intravital imaging procedures can be performed using basic surgery and animal maintenance techniques. More importantly, regular confocal microscopes - the same that are used for imaging immunofluorescence slides - can also acquire high quality intravital images and movies after minor adaptations. Here we propose minimal adaptations in stock microscopes that allow major improvements in different fields of scientific investigation.

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

Navigating challenges in the application of superresolution microscopy.

In 2014, the Nobel Prize in Chemistry was awarded to three scientists who have made groundbreaking contributions to the field of superresolution (SR) microscopy (SRM). The first commercial SR microscope came to market a decade earlier, and many other commercial options have followed. As commercialization has lowered the barrier to using SRM and the awarding of the Nobel Prize has drawn attention to these methods, biologists have begun adopting SRM to address a wide range of questions in many types of specimens. There is no shortage of reviews on the fundamental principles of SRM and the remarkable achievements made with these methods. We approach SRM from another direction: we focus on the current practical limitations and compromises that must be made when designing an SRM experiment. We provide information and resources to help biologists navigate through common pitfalls in SRM specimen preparation and optimization of image acquisition as well as errors and artifacts that may compromise the reproducibility of SRM data.

Impact of New Camera Technologies on Discoveries in Cell Biology.

New technologies can make previously invisible phenomena visible. Nowhere is this more obvious than in the field of light microscopy. Beginning with the observation of "animalcules" by Antonie van Leeuwenhoek, when he figured out how to achieve high magnification by shaping lenses, microscopy has advanced to this day by a continued march of discoveries driven by technical innovations. Recent advances in single-molecule-based technologies have achieved unprecedented resolution, and were the basis of the Nobel prize in Chemistry in 2014. In this article, we focus on developments in camera technologies and associated image processing that have been a major driver of technical innovations in light microscopy. We describe five types of developments in camera technology: video-based analog contrast enhancement, charge-coupled devices (CCDs), intensified sensors, electron multiplying gain, and scientific complementary metal-oxide-semiconductor cameras, which, together, have had major impacts in light microscopy.

Living Cells and Dynamic Molecules Observed with the Polarized Light Microscope: the Legacy of Shinya Inoué.

In 1948, Shinya Inoué arrived in the United States for graduate studies at Princeton. A year later he came to Woods Hole, starting a long tradition of summer research at the Marine Biological Laboratory (MBL), which quickly became Inoué's scientific home. Primed by his Japanese mentor, Katsuma Dan, Inoué followed Dan's mantra to work with healthy, living cells, on a fundamental problem (mitosis), with a unique tool set that he refined for precise and quantitative observations (polarized light microscopy), and a fresh and brilliant mind that was unafraid of challenging current dogma. Building on this potent combination, Inoué contributed landmark observations and concepts in cell biology, including the notion that there are dynamic, fine structures inside living cells, in which molecular assemblies such as mitotic spindle fibers exist in delicate equilibrium with their molecular building blocks suspended in the cytoplasm. In the late 1970s and 1980s, Inoué and others at the MBL were instrumental in conceiving video microscopy, a groundbreaking technique which married light microscopy and electronic imaging, ushering in a revolution in how we know and what we know about living cells and the molecular mechanisms of life. Here, we recount some of Inoué's accomplishments and describe how his legacy has shaped current activities in polarized light imaging at the MBL.

Utilidad del control por parte del patólogo del material obtenido por punción aspiración con aguja fina bajo guía de ecoendoscopia: revisión de la bibliografía acerca de un tema controvertido / Utility of rapid on-site evaluation (ROSE) by a pathologist during endoscopic ultrasound-guided fine needle aspiration cytology: a review of the literature

Numerosas técnicas de imagen permiten visualizar y obtener material de órganos de difícil acceso de forma mínimamente invasiva. Es preciso un estudio citológico adecuado, porque el calibre habitual de las agujas no permite obtener material histológico. Por ello, ligado al uso de la ecoendoscopia se debe hablar de punción aspiración con aguja fina (PAAF). La PAAF es una técnica económica cuando se realiza sobre lesiones palpables, pero con control por técnicas de imagen su rentabilidad es dudosa por la variabilidad entre centros y el aumento del coste. El objetivo de este estudio es determinar si el control por el patólogo del material obtenido en PAAF con ecoendoscopia mejora la precisión diagnóstica (en relación con la biopsia), cuál es la mejor estrategia para esta prueba, quién debe hacerla y si resulta económicamente rentable. Dada la escasez de datos a nivel nacional sobre este tema, hemos optado por una revisión bibliográfica de la literatura publicada (AU)

New imaging techniques, allowing the visualization and minimally invasive sampling of organs that are difficult to access, are being used more and more frequently. A suitable, accompanying cytological study is required, as in the majority of cases the caliber of the needle used is not sufficient to obtain a histological sample. Therefore, endoscopic ultrasound is generally associated with fine-needle aspiration (FNA). The performance of FNA on palpable nodules is a cost-effective procedure. However, the cost-effectiveness of using FNA with endoscopic ultrasound guidance is controversial, due to interobserver variability and increase in costs. The object of this study is to determinate if rapid-on site evaluation by a pathologist during endoscopic ultrasound-guided FNA improves diagnostic accuracy (correlated with biopsy), what is the best methodology, who should do it and the cost-effectiveness of the procedure. Because of the lack of Spanish data, we reviewed the pertinent international literature (AU)