Correlated cryogenic fluorescence microscopy and electron cryo-tomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo

Members of the tripartite motif (TRIM) protein family have been shown to assemble into structures in both the nucleus and cytoplasm. One TRIM protein family member, TRIM5α, has been shown to form cytoplasmic bodies involved in restricting retroviruses such as HIV-1. Here we applied cryogenic correlated light and electron microscopy, combined with electron cryo-tomography, to intact mammalian cells expressing YFP-rhTRIM5α and found the presence of hexagonal nets whose arm lengths were similar to those of the hexagonal nets formed by purified TRIM5α in vitro. We also observed YFP-rhTRIM5α within a diversity of structures with characteristics expected for organelles involved in different stages of macroautophagy, including disorganized protein aggregations (sequestosomes), sequestosomes flanked by flat double-membraned vesicles (sequestosome:phagophore complexes), sequestosomes within double-membraned vesicles (autophagosomes), and sequestosomes within multivesicular autophagic vacuoles (amphisomes or autolysosomes). Vaults were also seen in these structures, consistent with their role in autophagy. Our data 1) support recent reports that TRIM5α can form both well-organized signaling complexes and nonsignaling aggregates, 2) offer images of the macroautophagy pathway in a near-native state, and 3) reveal that vaults arrive early in macroautophagy

Development of cryogenic correlated light electron microscopy methods to study mechanisms of intracellular trafficking and their relationships to the secretory pathway

The application of cryogenic electron microscopy (cryo‐EM) to the study of cellular ultrastructure provides a resolution several orders of magnitude better than light microscopy. Although this approach is increasingly applied in situ, it suffers from limitations in our ability to target imaging to specific intracellular features including the subcellular localization of specific events of interest. Cryogenic correlated light and electron microscopy (cryo‐CLEM) helps to overcome this problem by spatially locating areas of interest inside cells using fluorescence from genetically tagged or stained cellular molecules and allows for the visualization of localized fluorescently‐tagged proteins down to the level of individual organelles. Here, we attempted to study the secretory pathway in a specialized mammalian cell line, insulin‐secreting INS‐1E cells, expressing genetically‐encoded fluorophores as a model system to develop a cryo‐CLEM methodology. We discovered that there are many bright sources of autofluorescence in frozen cells. Based on our initial observations and the current understanding in the field, we hypothesized that autofluorescence from endogenous cellular substrates exhibits a broader spectrum of fluorescence than the fluorescence range of our expressed fluorescent proteins. To test this, we developed a quantitative approach to discriminate between autofluorescence and the fluorescent signal from genetically‐encoded fluorophores by measuring fluorescent intensities across different bandwidths. To validate this new methodology, we visualized multiple fluorophore‐tagged organelle markers in our experimental cell system. We found that DsRed2‐cytochrome c oxidase and chromogranin A‐GFP proteins were targeted in INS‐1E cells to mitochondria and secretory granules by cryo‐CLEM, consistent with their respective well‐established intracellular localizations. Moreover, these fluorescent signals were clearly distinguishable from autofluorescence emanating from endogenous structures including insulin crystals and multilamellar bodies. Overall, our novel cryo‐CLEM methods open the door to the study of cellular phenomena and structures with a new degree of specificity

The impact of the winery's wastewater treatment system on the winery water footprint

In the Mediterranean region, water scarcity has already prompted concern in the wine sector due to the strong impact it has on vineyard productivity and wine quality. Water footprint is an indicator that takes account of all the water involved in the creation of a product and may help producers to identify hotspots, and reduce water consumption and the corresponding production costs. In recent years several studies have been reported on wine water footprint determination, but mostly focused on the viticulture phase or assuming no grey water footprint at the winery since it has a treatment system. In the framework of the WineWaterFootprint project a medium-size winery was monitored, with direct measurements, regarding determination of the blue and grey components of water footprint. The determined winery water footprint ranged from 9.6 to 12.7 L of water per wine bottle of 0.75 L, the wastewater produced being responsible for about 98%, which means that the grey component cannot be disregarded. The developed scenarios show that a potential reduction of 87% in winery water footprint can be obtained with almost no investment. The challenge of reducing the grey footprint is not in technology development, but rather in the proper maintenance and monitoring of treatment systemsinfo:eu-repo/semantics/publishedVersio

Correlated cryogenic fluorescence microscopy and electron cryo-tomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo

Members of the tripartite motif (TRIM) protein family have been shown to assemble into structures in both the nucleus and cytoplasm. One TRIM protein family member, TRIM5α, has been shown to form cytoplasmic bodies involved in restricting retroviruses such as HIV-1. Here we applied cryogenic correlated light and electron microscopy, combined with electron cryo-tomography, to intact mammalian cells expressing YFP-rhTRIM5α and found the presence of hexagonal nets whose arm lengths were similar to those of the hexagonal nets formed by purified TRIM5α in vitro. We also observed YFP-rhTRIM5α within a diversity of structures with characteristics expected for organelles involved in different stages of macroautophagy, including disorganized protein aggregations (sequestosomes), sequestosomes flanked by flat double-membraned vesicles (sequestosome:phagophore complexes), sequestosomes within double-membraned vesicles (autophagosomes), and sequestosomes within multivesicular autophagic vacuoles (amphisomes or autolysosomes). Vaults were also seen in these structures, consistent with their role in autophagy. Our data 1) support recent reports that TRIM5α can form both well-organized signaling complexes and nonsignaling aggregates, 2) offer images of the macroautophagy pathway in a near-native state, and 3) reveal that vaults arrive early in macroautophagy

Qualidade do milho doce minimamente processado conservado sob diferentes atmosferas.

Este trabalho teve por objetivo avaliar a influência de três atmosferas diferentes (2% O2 + 8% CO2, 4% O2 + 8% CO2 e atmosfera ambiente) na qualidade de milho verde do tipo doce minimamente processado, durante sua conservação a 5 °C. Foram utilizadas espigas de milho verde do tipo doce, híbrido do programa de melhoramento da Embrapa Milho e Sorgo com gene mutante sh2 (Embrapa HT1). O conteúdo de glicose, frutose, B-criptoxantina, firmeza e valor L* foram influenciadas somente pelo tempo de conservação. As atmosferas controladas foram eficientes em reduzir a perda de massa das espigas de milho doce e, pelos dados obtidos para o pH e acidez titulável, manteve a atividade respiratória reduzida em até três dias de armazenamento. O conteúdo de sacarose mais elevado nas espigas armazenadas em atmosfera ambiente pode, adicionalmente, indicar mais rápida degradação de amido e maior disponibilidade de substrato para a respiração. De modo geral, o uso da atmosfera controlada não influenciou nos teores de carotenóides totais. A atmosfera de 2% O2 e 8% CO2 apresentou maiores valores de b* e de firmeza. Todas as amostras de milho doce minimamente processado analisadas, independente das atmosferas de conservação, encontravam-se dentro dos limites microbiológicos aceitáveis especificados pela legislação. No entanto, a atmosfera de 2%O2 e 8%CO2 apresentou menores populações de coliformes a 35 °C, de bactérias aeróbias psicrotróficas e fungos filamentosos e leveduras, sendo, então, a mais indicada para a conservação pós-colheita dos milhos doces minimamente processados, durante nove dias

Distinguishing Signal From Autofluorescence In Cryogenic Correlated Light And Electron Microscopy Of Mammalian Cells

In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules. Here we describe a method to distinguish fluorescent protein tags from these autofluorescent sources based on the narrower emission spectrum of the former. The method is first tested on mitochondria and then applied to examine the ultrastructural variability of secretory granules within insulin-secreting pancreatic beta-cell-derived INS-1E cells

Current Trends in Functional Imaging of Pheochromocytomas and Paragangliomas

Most pheochromocytomas/paragangliomas should be evaluated with anatomical imaging (computed tomography or magnetic resonance imaging) followed by functional imaging (nuclear medicine modalities). Functional imaging assures that the tumor is indeed a pheochromocytoma/paraganglioma and enables more thorough localization, especially detecting as many lesions as possible (in particular for metastatic disease). Functional imaging for pheochromocytomas/paragangliomas, can use radiolabled ligands specific for pathways of synthesis, metabolism, and inactivation of catecholamines or nonspecific ligands. In an overview of the available nuclear medicine modalities, we summarize the accumulated experience and recommend when functional imaging should be applied to patients with pheochromocytoma/paraganglioma.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74881/1/annals.1353.041.pd

Myocardial perfusion reserve compared with peripheral perfusion reserve: A [13N]ammonia PET study

INTRODUCTION: [13N]ammonia PET allows quantification of myocardial perfusion. The similarity between peripheral flow and myocardial perfusion is unclear. We compared perfusion flow in the myocardium with the upper limb during rest and adenosine stress [13N]ammonia PET to establish whether peripheral perfusion reserve (PPR) correlates with MPR. METHODS: [13N]ammonia myocardial perfusion PET-scans of 58 patients were evaluated (27 men, 31 women, age 64 ± 13 years) and were divided in four subgroups: patients with coronary artery disease (CAD, n = 15), cardiac syndrome X (SX, n = 14), idiopathic dilating cardiomyopathy (DCM, n = 16), and normal controls (NC, n = 13). Peripheral limb perfusion was measured in the muscular tissue of the proximal upper limb and quantified through a 2-tissue-compartment model and the PPR was calculated (stress/rest ratio). MPR was also calculated by a 2-tissue-compartment model. The PPR results were compared with the MPR findings. RESULTS: Mean myocardial perfusion increased significantly in all groups as evidenced by the MPR (CAD 1.99 ± 0.47; SX 1.39 ± 0.31; DCM 1.72 ± 0.69; NC 2.91 ± 0.78). Mean peripheral perfusion also increased but not significantly and accompanied with great variations within and between groups (mean PPR: CAD 1.30 ± 0.79; SX 1.36 ± 0.71; DCM 1.60 ± 1.22; NC 1.27 ± 0.63). The mean difference between PPR and MPR for all subpopulations varied widely. No significant correlations in flow reserve were found between peripheral and myocardial microcirculatory beds in any of the groups (Total group: r = -0.07, SEE = 0.70, CAD: r = 0.14, SEE = 0.48, SX: r = 0.17, SEE = 0.30, DCM: r = -0.11, SEE = 0.71, NC: r = -0.19, SEE = 0.80). CONCLUSION: No correlations between myocardial and peripheral perfusion (reserve) were found in different patient populations in the same PET session. This suggests a functional difference between peripheral and myocardial flow in the response to intravenously administered adenosine stress