Transmission electron microscopy (TEM) is widely used as an imaging modality to provide high-resolution details of subcellular components within cells and tissues. Mitochondria and endoplasmic ...reticulum (ER) are organelles of particular interest to those investigating metabolic disorders. A straightforward method for quantifying and characterizing particular aspects of these organelles would be a useful tool. In this protocol, we outline how to accurately assess the morphology of these important subcellular structures using open source software
, originally developed by the National Institutes of Health (NIH). Specifically, we detail how to obtain mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology and measuring mito/endoplasmic reticulum (ER) interactions. These procedures provide useful tools for quantifying and characterizing key features of sub-cellular morphology, leading to accurate and reproducible measurements and visualizations of mitochondria and ER.
High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging ...cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.
Transmembrane proteins (TMEMs) are integral proteins that span biological membranes. TMEMs function as cellular membrane gates by modifying their conformation to control the influx and efflux of ...signals and molecules. TMEMs also reside in and interact with the membranes of various intracellular organelles. Despite much knowledge about the biological importance of TMEMs, their role in metabolic regulation is poorly understood. This review highlights the role of a single TMEM, transmembrane protein 135 (TMEM135). TMEM135 is thought to regulate the balance between mitochondrial fusion and fission and plays a role in regulating lipid droplet formation/tethering, fatty acid metabolism, and peroxisomal function. This review highlights our current understanding of the various roles of TMEM135 in cellular processes, organelle function, calcium dynamics, and metabolism.
Watershed managers generally focus on P reduction strategies to combat freshwater eutrophication despite evidence that N co‐limits primary production. Our objective was to test the role of P in ...limiting stream periphyton biomass within the Buffalo River watershed in Arkansas by conducting a 31‐d streamside mesocosm experiment. To represent potentially different starting states, cobbles were transplanted from two different tributary streams and initially exposed to a range of P (0, 0.012, 0.025, 0.05, 0.1, and 0.2 mg L−1 P) to assess benthic ash‐free dry mass (AFDM) and chlorophyll‐a (chl a) and responses during a P only enrichment period. Later, the experiment was continued under a N/P (10:1 molar ratio) enrichment gradient to examine co‐limitation. Mean AFDM was higher on Day 31 of the N+P enrichment compared with Day 17 of the P‐only enrichment (p < .001). Overall differences in AFDM and chl a were observed between cobbles from different stream sites. Phosphorus enrichment stimulated benthic chl a biomass, but enrichment effects were greater when streams were enriched with N+P (p < .001). Chlorophyll‐a increased (4.4–57.9 mg m−2) with increasing P concentrations (p < .001) after P enrichment but was threefold greater after N+P enrichment, increasing from 13.3 to 171.1 mg m−2 across the enrichment gradient. Results support the need to consider both N and P limitation in freshwater systems and demonstrate that potential increases in nutrient concentrations may influence accumulation of algae on cobble substrates from the Buffalo River watershed.
Migration to the United States of America from Guatemala effects many aspects of Guatemalan life. We document, through extensive ethnographic fieldwork, how migrants and their remittances effect ...gender relations, ethnicity, land use, and land distribution. Our evidence is drawn from research in four communities. San Pedro Pinula and Gualán represent communities of eastern Guatemala. San Cristóbal Totonicapán is an Indigenous town in Guatemala’s western highlands, and San Lucas is a lowland frontier community in the Guatemalan department of Ixcán, which borders Chiapas, Mexico. Our results reveal that migrants and their remittances, both social and tangible, result in significant changes in land use and land distribution in Ixcán. Migrant money permits the conversion of rainforest into cattle pasture and also results in the accumulation of land in the hands of migrants. In terms of land use, we see in San Pedro Pinula that migrant money also allows the Pokoman Maya to make small entries into the Ladino (non-indigenous) dominated cattle business. In San Pedro Pinula, the migration and return of Maya residents also permits them to slowly challenge ethnic roles that have developed over the last five centuries. When we look at how migration effects gender roles in Gualán and San Cristóbal we also note that migration and social remittances permit a gradual challenge and erosion of traditional gender roles in Guatemala. We point out, however, that migration-related changes to traditional gender and ethnic roles is gradual because migrants, despite their increased earnings and awareness, run into a social structure that resists rapid change. This is not the case when we examine land transformations in Ixcán. Here, migrants encounter few barriers when they attempt to put their new money and ideas to work. Despite the advantages that migration brings to many families, especially in the face of a faltering national economy and state inactivity regarding national development, we conclude that migration and remittances do not result in community or nation-wide development. At this stage migrant remittances are used for personal advancement and very little money and effort is invested in works that benefit communities or neighborhoods. We call for continued studies of the effects of international migration on Guatemalan hometowns that build on our initial studies to better understand the longer-term ramifications of migration in a country where no community is without migrants.
To determine if mitochondrial pyruvate transport could represent a therapeutic target for sensitizing cancer cells to oxidative stress, lung and breast cancer cells were treated with 5 µM UK5099 to ...inhibit the mitochondrial pyruvate carrier (MPC). Treatment with UK5099 selectively sensitized lung and breast cancer cells to clonogenic cell killing when combined with depletion of glutathione using 1 mM buthionine sulfoximine (BSO; a glutathione synthesis inhibitor) for 48 h and 72 h, relative to normal lung and breast epithelial cells. Furthermore, cancer cell killing mediated by UK5099 combined with BSO was inhibited by the thiol antioxidant, N-acetylcysteine (NAC; 20 mM), independent of GSH levels indicating a mechanism of toxicity involving reduced thiols and metabolic oxidative stress. In addition, treatment with UK5099 alone for 48 h also decreased levels of total glutathione in cancer cells that could be reversed by NAC. Using oxidation sensitive fluorescent dyes (CDCFH2 and MitoSOX), treatment of lung and breast cancer cells for 24 h and 48 h with UK5099 induced increases in steady state levels of pro-oxidants (presumably hydroperoxides and mitochondrial superoxide) which were further increased with BSO. Finally, treatment of breast cancer cells with UK5099 for 24 and 48 hours significantly sensitized breast cancer cells to clonogenic cell killing mediated by paclitaxel. These data support the hypothesis that inhibition of the MPC selectively causes an impairment of antioxidant capability in cancer cells that is enhanced by depletion of glutathione. Furthermore, these results also support the hypothesis that inhibition of the MPC represents a significant target for sensitizing human breast cancer cells to chemotherapy agents thought to induce oxidative stress.
The Cornea Donor Study (CDS) showed that donor age is not a factor in survival of most penetrating keratoplasties for endothelial disease. Secondary analyses confirm the importance of surgical ...indication and presence of glaucoma in outcomes at 10 years.
To assess the relationship between donor and recipient factors and corneal graft survival in the CDS.
Multicenter prospective, double-masked, controlled clinical trial conducted at 80 clinical sites. One hundred five surgeons enrolled 1090 participants undergoing corneal transplant for a moderate-risk condition, principally Fuchs dystrophy or pseudophakic or aphakic corneal edema (PACE). Forty-three eye banks provided corneas.
Corneas from donors younger than 66 years and donors 66 years or older were assigned, masked to donor age. Surgery and postoperative care were performed according to the surgeons' usual routines. Participants were followed up for as long as 12 years.
Graft failure, defined as a regrafting procedure or a cloudy cornea for 3 consecutive months.
The 10-year cumulative probability of graft failure was higher in participants with PACE than in those with Fuchs dystrophy (37% vs 20%; hazard ratio HR, 2.1 99% CI, 1.4-3.0; P < .001) and in participants with a history of glaucoma before penetrating keratoplasty, particularly with prior glaucoma surgery (58% with prior glaucoma surgery and use of medications to lower intraocular pressure at the time of surgery vs 22% with no history of glaucoma surgery or medication use; HR, 4.1 99% CI, 2.2-7.5; P < .001). We found trends toward increased graft failure in recipients who were 70 years or older compared with those younger than 60 years (29% vs 19%; HR, 1.2 99% CI, 0.7-2.1; P = .04) or were African American (HR, 1.5; P = .11) or who had a history of smoking (35% vs 24%; HR, 1.6 99% CI, 0.9-2.8; P = .02). Lower endothelial cell density (ECD) and higher corneal thickness (CT) at 6 months (6% vs 41% for ECD ≥2700 vs <1700 cells/mm2 P < .001; 14% vs 36% for CT <500 vs ≥600 μm P = .001), 1 year (4% vs 39% for ECD ≥2700 vs <1700 cells/mm2 P < .001; 18% vs 28% for CT <500 vs ≥600 μm P = .04), and 5 years (2% vs 29% for ECD ≥1500 vs <500 cells/mm2 P < .001; 7% vs 34% for CT <550 vs ≥650 μm P < .001) were associated with subsequent graft failure.
Most penetrating corneal grafts for Fuchs dystrophy or PACE remain clear at 10 years. The risk for failure is greater for graft recipients with PACE and those with a history of glaucoma. Measurements of ECD and CT during the course of postkeratoplasty follow-up are associated with a risk for failure. However, even with very low ECD and high CT at 5 years, most corneas remain clear at 10 years.
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