Fixed-angle Rotor Research Articles

A-108 Validation of Plasma Oxalate Measurement on the Roche Cobas c501

Abstract Background Measurement of plasma oxalate is important in patients with renal failure as oxalate may build up due to impaired excretion and lead to organ damage due to precipitation of calcium oxalate crystals. However, the measurement of plasma oxalate is challenging due to the low concentrations present in individuals with healthy kidney function and the pre-analytical steps needed to prevent erroneous oxalate results due to conversion from ascorbate. In order to provide improved turn-around-time for plasma oxalate testing, our objective was to develop and validate a method to measure plasma oxalate on the Roche cobas c501. Methods Samples used in the validation study included both fresh collections and residual plasma from specimens collected in lithium heparin, no-gel tubes. Samples from fresh collections were kept on ice prior to centrifugation, and then frozen if not analyzed immediately. Thawed plasma was acidified by addition of 12.1 M HCl to a pH between 2 and 3 and mixed thoroughly by vortexing. One milliliter of the acidified plasma was added to a Centrifree filter (Millipore). The samples were then centrifuged for 90 min at 1500 × g 20°C in a fixed angle rotor to remove plasma proteins. The resulting filtrate was assayed on the Roche cobas c501. The assay (Trinity Biotech) uses oxalate oxidase to produce an indamine dye through the production of hydrogen peroxide from oxalate in the sample, which is measured spectrophotometrically at 600 nm at a single time point. The analytical measuring range (AMR) was evaluated by performing serial dilutions of a high sample. Interferences from hemolysis, icterus, and lipemia were assessed at both low and high oxalate concentrations. Precision was evaluated using two QC concentrations, and patient samples with low and high concentrations of oxalate. Samples were tested ten times in a single day, or twice per day for ten days to estimate intra- and inter-precision, respectively. Spike recovery studies were used to assess method accuracy. Lastly, reference intervals were verified by assaying samples from twenty healthy volunteers. Total allowable error was ±30% or 2 µmol/L with a maximum 15%CV. Results The assay was validated to be linear across an AMR of 2 to 30 µmol/L, with an average recovery of 105% across five concentrations. The total assay imprecision was <15%. Initial spike recovery studies across the AMR showed an average recovery of 99.4% (n = 12), with a range of 76.6 to 132.4%. In a mixing study, hemolyzed and lipemic sample recovery was on average 82.1% and 76.1% respectively, which was low, but acceptable. Samples mixed with icteric specimens demonstrated unacceptably low recovery (average of 64.1%). The reference interval of <2 µmol/L was successfully verified as all twenty healthy volunteers had results <2 µmol/L. Conclusion Initial validation studies support the clinical utility of the newly developed method. Plasma oxalate methods are documented to have significant inter-method variability and variable recovery, which is hypothesized to be due to variation at the filtration step. This semi-automated assay will require less technologist time and will reduce the possibility for human error affecting the result.

Aerodynamic Performance and Wake Characteristics Analysis of Archimedes Spiral Wind Turbine Rotors with Different Blade Angle

Continuous improvement of wind turbines represent an effective way of achieving green energy and reducing dependence on fossil fuel. Conventional lift-type horizontal axis wind turbines, which are widely used, are designed to run under high wind speed to obtain a high efficiency. Aiming to use the low-speed wind in urban areas, a novel turbine, which is called the Archimedes Spiral Wind Turbine (abbreviated as ASWT), was recently proposed for low-speed wind applications. In the current work, a numerical simulation on the five ASWT rotors with different blade angles was carried out, which were performed to predict the detailed aerodynamic performance and wake characteristics. The results show that the ASWT rotor with a large blade angle has a wider operating tip speed ratio range and a higher tip speed ratio point of maximum power coefficient within a certain range, and yet the ASWT rotor with the larger blade angle has a higher thrust coefficient. Additionally, the ASWT rotor with a large blade angle usually has a large power coefficient and thrust coefficient fluctuation amplitude. On the other hand, the ASWT rotor with a small blade angle permits the undisturbed free stream to pass through the rotor blades more easily than that with a large blade angle. This causes a stronger blockage effect for the ASWT rotor with a large blade angle. Moreover, the blade angle also has a great effect on the shape of the vortex structure. The blade tip vortex of the fixed-angle ASWT rotors is more stable than those of the variable-angle ASWT rotors. The hub vortex of the ASWT rotors with a large blade angle is stronger than those with a small blade angle. Meanwhile, the wake recovery for ASWT rotors with a small blade angle is evidently lower than those with a large blade angle.

White-flesh guava juice clarification by a fixed-angle conical rotor centrifuge laboratory and characterization of continuous disk stack centrifuges

The two objectives of this paper were to determine the effect of centrifugation parameters on guava juice physicochemical characteristics and to identify operational characteristics of continuous disc-stack centrifuges based on the performance of a laboratory centrifuge. Effects of g-force (149 g–3731 g) and centrifugation time (10–40 min) on juice physicochemical characteristics (protein, pectin, galacturonic acid, dry matter, total soluble sugars contents; pH; electrical conductivity, clarity and particle size distribution) were assessed. Laboratory centrifuge performance was evaluated for 1343 g, 2388 g and 3731 g. At 1343 g, separation limits (xmax), feed flow-rates of disc-stack centrifuges and cut-off sizes (x50) were determined for corresponding laboratory centrifuge operation times. Significant decrease in average particle size, protein and pectin contents was observed, contrary to juice clarity. Similar clarification efficiency was obtained for g-forces ≥ 1343 g. xmax were 2669 nm, 2200 nm and 1783 nm, with corresponding x50 for continuous centrifuges, 1887 nm, 1556 nm, 1261 nm, for 20 min, 30 min and 40 min, respectively.

A Standardized g-Force Allows the Preparation of Similar Platelet-Rich Fibrin Qualities Regardless of Rotor Angle.

Platelet-rich fibrin (PRF) is an autologous blood concentrate that supports tissue regeneration. The effect of the centrifuge rotor angle in the fabrication of PRF is still not fully elucidated. The hypothesis of this study is: When applying the same g-force (relative centrifugal force [RCF]) and centrifugation time, PRF components and bioactivity are not modified using either a swing-out rotor or a fixed angle rotor. For this purpose, peripheral blood samples (from five donors) were used to gain solid (710 ×g, 8 min) and liquid (44 ×g, 8 min) PRF matrices using three different centrifuges (one fixed angle as a control and two different swing-out rotor centrifuges). The physical characteristics of the solid PRF were measured to evaluate the clot formation and cellular distribution. The liquid PRF was used to evaluate the cell number, bioactivity, and influence on primary human osteoblasts (pOBs) and primary human fibroblasts (pHFs) in vitro. Solid PRF clots were significantly larger in the group of fixed rotor centrifuges compared with either of the two evaluated swing-out rotor centrifuges. No differences were observed when evaluating the cellular distribution within the solid PRF. No statistically significant differences were documented in the cell's density in liquid PRF samples (platelets, lymphocytes, neutrophils, eosinophils, and basophils) among the differently gained PRF samples. No statistically significant differences were documented for the released growth factors (vascular endothelial growth factor, epidermal growth factor, and transforming growth factor beta 1) over 7 days. pOBs and pHFs viability after treatment with PRF conditioned media showed no statistically significant differences between the evaluated groups. However, the number of adherent cells treated with PRF obtained with the use of the fixed angle rotor was significantly higher when compared with those treated with PRF obtained by using the swing-out rotors. The presented results confirm that regardless of the centrifuge rotor used, the components and bioactivity of solid and liquid PRF matrices are modified by the applied RCF and centrifugation time. These findings are of great importance for highlighting the essential role of adapting the centrifugation protocols when using different centrifuges and to correctly report the used centrifugation protocols in scientific research to allow for reproducible results. Impact statement Platelet-rich fibrin (PRF) is prepared from autologous peripheral blood and is widely applied in research and clinical treatments. The centrifugation parameters used during the preparation of PRF directly influence its components and bioactivity. By using a standardized protocol, the present study demonstrated that adapting various centrifuges to a standardized relative centrifugal force and centrifugation protocol resulted in reproducible PRF matrices with similar bioactivity, regardless of the centrifuge rotor angle. These findings underline the necessity to carefully adapt and correctly report the used centrifuge and centrifugation protocols in scientific research to allow reproducible results.

Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

A system for obtaining soil solution extracts and soil water retention curves using a bench centrifuge with fixed angle rotors

A centrifugation system is proposed for recovering soil solution extracts from disturbed natural soils. The water retention curve (WRC), which is related to the hydraulic properties of soil, was determined as a way to define the range of suctions applied for the purpose of soil water extraction. The design of the centrifugation device, the analysis of the experimental procedure and its validation are the aims of this work. Since centrifugation time is an important parameter for water extraction, a systematic study was performed. Although very long centrifugation times are generally required to guarantee equilibrium is reached, a time period of 60 min was used in the tests. The van Genuchten model was fitted to the experimental results of retained moisture versus effective suction, representing the matric potential and achieved thru the angular velocity of the rotor. The results obtained by this new approach were compared with the results obtained using the pressure chamber. This comparison revealed a systematic reduction in the amount of solution extracted at high suctions (above 100 kPa) regarding the pressure chamber. A feedback effect was confirmed by using ceramic sintered-perlite beads instead of sintered glass beads to support the soil specimen in the device adapted to the centrifuge. The proposed system was able to extract soil solutions in a fast, repeatable, and simple way.

Note: Rotor design optimization for vacuum chamber-free ultracentrifuge.

This paper proposes a fixed-angle rotor design for a vacuum chamber-free ultracentrifuge using a rigid shaft and magnetic bearings. The rotor is designed for a stable operation in the ultracentrifugation region with air friction. And the optimal design parameters and optimized connection method of the rotor and rigid shaft are proposed. The proposed rotor structure can withstand a maximum rotation speed of 100 000 rpm and achieves a maximum relative centrifugal force of 600 000 g. To verify the performance of the proposed rotor design with a rigid shaft, finite-element analysis, an impact test, and a drive operating experiment were conducted.

The in vitro release of cytokines and growth factors from fibrin membranes produced through horizontal centrifugation.

Platelet-rich fibrin membranes are biomaterials widely used for therapeutic purposes, and canonically produced through the processing of peripheral blood with fixed-angle rotor centrifuges. In this work, we evaluate the in vitro stability and release of cytokines and growth factors when these biomaterials are produced with a horizontal swing-out clinical centrifuge. Membranes produced from the blood of 14 donors were morphologically evaluated by scanning electron microscopy and fluorescence microscopy, and their stability was assessed by photographic recording after incubation in culture medium for up to 28 days. The release of 27 cytokines and growth factors was monitored for three weeks through a multiparametric immunoassay. The fibrin membranes presented complex three-dimensional structure with a high density of nucleated cells. A large release of growth factors [platelet derived growth factor, fibroblastic growth factor (bFGF), and vascular endothelial growth factor] was detected in the first 24 h, followed by time-dependent decay, maintaining significant concentrations after three weeks. Both anti-inflammatory and pro-inflammatory cytokines presented different release peaks, maintaining high rates of elution for up to 21 days. Chemokines of relevance in tissue repair [RANTES, granulocyte colony-stimulating factor (G-CSF)] were also produced in large quantities throughout the experimental period. The present results demonstrate that blood-derived fibrin membranes with high structural stability and cell content can be generated by horizontal centrifugation, being able of a prolonged production/release of growth factors and pro- and anti-inflammatory cytokines. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1373-1380, 2018.

Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations

Diagnostic methods that focus on the extracellular vesicles (EVs) present in saliva have been attracting great attention because of their non-invasiveness. EVs contain biomolecules such as proteins, messenger RNA (mRNA) and microRNA (miRNA), which originate from cells that release EVs, making them an ideal source for liquid biopsy. Although there have been many reports on density-based fractionation of EVs from blood and urine, the number of reports on EVs from saliva has been limited, most probably because of the difficulties in separating EVs from viscous saliva using density gradient centrifugation. This article establishes a protocol for the isolation of EVs from human saliva using density gradient centrifugation. The fractionated salivary EVs were characterized by atomic force microscopy, western blot and reverse transcription polymerase chain reaction. The results indicate that salivary EVs have a smaller diameter (47.8±12.3 nm) and higher density (1.11 g/ml) than EVs isolated from conditioned cell media (74.0±23.5 nm and 1.06 g/ml, respectively). Additionally, to improve the throughput of density-based fractionation of EVs, the original protocol was further modified by using a fixed angle rotor instead of a swinging rotor. It was also confirmed that several miRNAs were expressed strongly in the EV-marker-expressing fractions.

The GlycoFilter: A Simple and Comprehensive Sample Preparation Platform for Proteomics, N-Glycomics and Glycosylation Site Assignment

Current strategies to study N-glycoproteins in complex samples are often discrete, focusing on either N-glycans or N-glycosites enriched by sugar-based techniques. In this study we report a simple and rapid sample preparation platform, the GlycoFilter, which allows a comprehensive characterization of N-glycans, N-glycosites, and proteins in a single workflow. Both PNGase F catalyzed de-N-glycosylation and trypsin digestions are accelerated by microwave irradiation and performed sequentially in a single spin filter. Both N-glycans and peptides (including de-N-glycosylated peptides) are separately collected by filtration. The condition to effectively collect complex and heterogeneous N-glycans was established on model glycoproteins, bovine ribonuclease B, bovine fetuin, and human serum IgG. With this platform, the N-glycome, N-glycoproteome and proteome of human urine and plasma were characterized. Overall, a total of 865 and 295 N-glycosites were identified from three pairs of urine and plasma samples, respectively. Many sites were defined unambiguously as partially occupied by the detection of their nonsugar-modified peptides (128 from urine and 61 from plasma), demonstrating that partial occupancy of N-glycosylation occurs frequently. Given the likely high prevalence and variability of partial occupancy, glycoprotein quantification based exclusively on deglycosylated peptides may lead to inaccurate quantification.

BCL-2 Is a Downstream Target of ATF5 That Mediates the Prosurvival Function of ATF5 in a Cell Type-dependent Manner

ATF5 loss of function has been shown previously to cause apoptotic cell death in glioblastoma and breast cancer cells but not in non-transformed astrocytes and human breast epithelial cells. The mechanism for the cell type-dependent survival function of ATF5 is unknown. We report here that the anti-apoptotic factor BCL-2 is a downstream target of ATF5 that mediates the prosurvival function of ATF5 in C6 glioma cells and MCF-7 breast cancer cells. ATF5 binds to an ATF5-specific regulatory element that is downstream of and adjacent to the negative regulatory element in the BCL-2 P2 promoter, stimulating BCL-2 expression. Highlighting the critical role of BCL-2 in ATF5-dependent cancer cell survival, expression of BCL-2 blocks death of C6 and MCF-7 cells induced by dominant-negative ATF5, and depletion of BCL-2 impairs ATF5-promoted cell survival. Moreover, we found that BCL-2 expression is not regulated by ATF5 in non-transformed rat astrocytes, mouse embryonic fibroblasts, and human breast epithelial cells, where expression of BCL-2 but not ATF5 is required for cell survival. These findings identify BCL-2 as an essential mediator for the cancer-specific cell survival function of ATF5 in glioblastoma and breast cancer cells and provide direct evidence that the cell type-specific function of ATF5 derives from differential regulation of downstream targets by ATF5 in different types of cells.

Molecular Biology Techniques Q&A

Molecular Biology Techniques Q&A

ORIGINAL RESEARCH: Centrifugal recovery of rhizobial cells from fermented starch industry wastewater & development of stable formulation

Abstract Application of rhizobial inoculant is an agricultural practice successfully used to increase growth and yield of leguminous plants. Development of a concentrated culture is essential for advanced formulations. This study assessed the use of starch industry wastewater as a potent carbon source for production of Sinorhizobium meliloti, with a maximum cell count of 4×109 CFU/mL. Optimal conditions for maximum recovery of cells (>99%) during centrifugation were determined to be 8 000 g, at a temperature of 20 °C for 20 min, pH 7, using a swinging bucket rotor and a surface response methodology. Of the different centrifugal aids tested, starch (2% w/v) best minimized the loss of microbial cells during recovery from the supernatant. A fixed- angle-rotor experiment was also carried out to determine differences in recovery between centrifugal configurations; optimal recovery was observed with the swinging bucket rotor (>99%) versus a fixed angle rotor (90%). A further decrease (from 90% to 87%) in recove...

Chronic kidney disease delays VLDL-apoB-100 particle catabolism: potential role of apolipoprotein C-III

To determine the relative contribution of obesity and/or insulin resistance (IR) in the development of dyslipidemia in chronic kidney disease (CKD), we investigated the transport of apolipoprotein (apo) B-100 in nonobese, nondiabetic, nonnephrotic CKD subjects and healthy controls (HC). We determined total VLDL, VLDL(1), VLDL(2), intermediate density lipoprotein (IDL), and LDL-apoB-100 using intravenous D3-leucine, GC-MS, and multicompartmental modeling. Plasma apoC-III and apoB-48 were immunoassayed. In this case control study, we report higher plasma triglyceride, IDL-, VLDL-, VLDL(1)-, and VLDL(2)-apoB-100 concentrations in CKD compared with HC (P < 0.05). This was associated with decreased fractional catabolic rates [FCRs (pools/day)] [IDL:CKD 3.4 (1.6) vs. HC 5.0 (3.2), P < 0.0001; VLDL:CKD 4.8 (5.2) vs. HC 7.8 (4.8), P = 0.038; VLDL(1):CKD 10.1 (8.5) vs. HC 29.5 (45.1), P = 0.007; VLDL(2):CKD 5.4 (4.6) vs. HC 10.4 (3.4), P = 0.001] with no difference in production rates. Plasma apoC-III and apoB-48 were significantly higher in CKD (P < 0.001) and both correlated with impaired FCRs of VLDL, VLDL(1), and VLDL(2) apoB-100 (P < 0.05). In CKD, apoC-III concentration was the only independent predictor of clearance defects in VLDL and its subfractions. Moderate CKD in the absence of central adiposity and IR is associated with mild hypertriglyceridemia due to delayed catabolism of triglyceride rich lipoproteins, IDL, and VLDL, without changes in production rate. Altered apoC-III metabolism may contribute to dyslipidemia in CKD, and this requires further investigation.

Lipid Rafts and Caveolin-1 Coordinate Interleukin-1β (IL-1β)-dependent Activation of NFκB by Controlling Endocytosis of Nox2 and IL-1β Receptor 1 from the Plasma Membrane

Recent evidence suggests that signaling by the proinflammatory cytokine interleukin-1beta (IL-1beta) is dependent on reactive oxygen species derived from NADPH oxidase. Redox signaling in response to IL-1beta is known to require endocytosis of its cognate receptor (IL-1R1) following ligand binding and the formation of redox-active signaling endosomes that contain Nox2 (also called redoxosomes). The consequent generation of reactive oxygen species by redoxosomes is responsible for the downstream recruitment of IL-1R1 effectors (IRAK, TRAF6, and IkappaB kinase kinases) and ultimately for activation of the transcription factor NFkappaB. Despite this knowledge of the signaling events that occur downstream of redoxosome formation, an understanding of the mechanisms that coordinate the genesis of redoxosomes following IL-1beta stimulation has been lacking. Here, we demonstrate that lipid rafts play an important role in this process. We show that Nox2 and IL-1R1 localize to plasma membrane lipid rafts in the unstimulated state and that IL-1beta signals caveolin-1-dependent endocytosis of both proteins into the redoxosome. We also show that inhibiting lipid raft-mediated endocytosis prevents NFkappaB activation. Finally, we demonstrate that Vav1, a Rac1 guanine exchange factor and activator of Nox2, is recruited to lipid rafts following IL-1beta stimulation and that it is required for NFkappaB activation. Our results fill in an important mechanistic gap in the understanding of early IL-1R1 and Nox2 signaling events that control NFkappaB activation, a redox-dependent process important in inflammation.

Hermansky-Pudlak Syndrome Protein Complexes Associate with Phosphatidylinositol 4-Kinase Type II α in Neuronal and Non-neuronal Cells

The Hermansky-Pudlak syndrome is a disorder affecting endosome sorting. Disease is triggered by defects in any of 15 mouse gene products, which are part of five distinct cytosolic molecular complexes: AP-3, homotypic fusion and vacuole protein sorting, and BLOC-1, -2, and -3. To identify molecular associations of these complexes, we used in vivo cross-linking followed by purification of cross-linked AP-3 complexes and mass spectrometric identification of associated proteins. AP-3 was co-isolated with BLOC-1, BLOC-2, and homotypic fusion and vacuole protein sorting complex subunits; clathrin; and phosphatidylinositol-4-kinase type II alpha (PI4KIIalpha). We previously reported that this membrane-anchored enzyme is a regulator of AP-3 recruitment to membranes and a cargo of AP-3 ( Craige, B., Salazar, G., and Faundez, V. (2008) Mol. Biol. Cell 19, 1415-1426 ). Using cells deficient in different Hermansky-Pudlak syndrome complexes, we identified that BLOC-1, but not BLOC-2 or BLOC-3, deficiencies affect PI4KIIalpha inclusion into AP-3 complexes. BLOC-1, PI4KIIalpha, and AP-3 belong to a tripartite complex, and down-regulation of either PI4KIIalpha, BLOC-1, or AP-3 complexes led to similar LAMP1 phenotypes. Our analysis indicates that BLOC-1 complex modulates the association of PI4KIIalpha with AP-3. These results suggest that AP-3 and BLOC-1 act, either in concert or sequentially, to specify sorting of PI4KIIalpha along the endocytic route.

Regulatory effects of arachidonate 5-lipoxygenase on hepatic microsomal TG transfer protein activity and VLDL-triglyceride and apoB secretion in obese mice

As 5-lipoxygenase (5-LO) is an emerging target in obesity and insulin resistance, we have investigated whether this arachidonate pathway is also implicated in the progression of obesity-related fatty liver disease. Our results show that 5-LO activity and 5-LO-derived product levels are significantly elevated in the liver of obese ob/ob mice with respect to wild-type controls. Treatment of ob/ob mice with a selective 5-LO inhibitor exerted a remarkable protection from hepatic steatosis as revealed by decreased oil red-O staining and reduced hepatic triglyceride (TG) concentrations. In addition, 5-LO inhibition in ob/ob mice downregulated genes involved in hepatic fatty acid uptake (i.e., L-FABP and FAT/CD36) and normalized peroxisome proliferator-activated receptor alpha (PPARalpha) and acyl-CoA oxidase expression, whereas the expression of lipogenic genes [i.e., fatty acid synthase (FASN) and SREBP-1c] remained unaltered. Furthermore, 5-LO inhibition restored hepatic microsomal TG transfer protein (MTP) activity in parallel with a stimulation of hepatic VLDL-TG and apoB secretion in ob/ob mice. Consistent with these findings, 5-LO products directly inhibited MTP activity and triggered cytosolic TG accumulation in CC-1 cells, a murine hepatocyte cell line. Taken together, these findings identify a novel steatogenic role for 5-LO in the liver through mechanisms involving the regulation of hepatic MTP activity and VLDL-TG and apoB secretion.

Hhat Is a Palmitoylacyltransferase with Specificity for N-Palmitoylation of Sonic Hedgehog

Palmitoylation of Sonic Hedgehog (Shh) is critical for effective long- and short-range signaling. Genetic screens uncovered a potential palmitoylacyltransferase (PAT) for Shh, Hhat, but the molecular mechanism of Shh palmitoylation remains unclear. Here, we have developed and exploited an in vitro Shh palmitoylation assay to purify Hhat to homogeneity. We provide direct biochemical evidence that Hhat is a PAT with specificity for attaching palmitate via amide linkage to the N-terminal cysteine of Shh. Other palmitoylated proteins (e.g. PSD95 and Wnt) are not substrates for Hhat, and Porcupine, a putative Wnt PAT, does not palmitoylate Shh. Neither autocleavage nor cholesterol modification is required for Shh palmitoylation. Both the Shh precursor and mature protein are N-palmitoylated by Hhat, and the reaction occurs during passage through the secretory pathway. This study establishes Hhat as a bona fide Shh PAT and serves as a model for understanding how secreted morphogens are modified by distinct PATs.

Peroxisomes Contain a Specific Phytanoyl-CoA/Pristanoyl-CoA Thioesterase Acting as a Novel Auxiliary Enzyme in α- and β-Oxidation of Methyl-branched Fatty Acids in Mouse

Phytanic acid and pristanic acid are derived from phytol, which enter the body via the diet. Phytanic acid contains a methyl group in position three and, therefore, cannot undergo beta-oxidation directly but instead must first undergo alpha-oxidation to pristanic acid, which then enters beta-oxidation. Both these pathways occur in peroxisomes, and in this study we have identified a novel peroxisomal acyl-CoA thioesterase named ACOT6, which we show is specifically involved in phytanic acid and pristanic acid metabolism. Sequence analysis of ACOT6 revealed a putative peroxisomal targeting signal at the C-terminal end, and cellular localization experiments verified it as a peroxisomal enzyme. Subcellular fractionation experiments showed that peroxisomes contain by far the highest phytanoyl-CoA/pristanoyl-CoA thioesterase activity in the cell, which could be almost completely immunoprecipitated using an ACOT6 antibody. Acot6 mRNA was mainly expressed in white adipose tissue and was co-expressed in tissues with Acox3 (the pristanoyl-CoA oxidase). Furthermore, Acot6 was identified as a target gene of the peroxisome proliferator-activated receptor alpha (PPARalpha) and is up-regulated in mouse liver in a PPARalpha-dependent manner.

Aup1p, a Yeast Mitochondrial Protein Phosphatase Homolog, Is Required for Efficient Stationary Phase Mitophagy and Cell Survival

Autophagy is a catabolic membrane-trafficking process that occurs in all eukaryotic cells and leads to the hydrolytic degradation of cytosolic material in the vacuolar or lysosomal lumen. Mitophagy, a selective form of autophagy targeting mitochondria, is poorly understood at present. Several recent reports suggest that mitophagy is a selective process that targets damaged mitochondria, whereas other studies imply a role for mitophagy in cell death processes. In a screen for protein phosphatase homologs that functionally interact with the autophagy-dedicated protein kinase Atg1p in yeast, we have identified Aup1p, encoded by Saccharomyces cerevisiae reading frame YCR079w. Aup1p is highly similar to a family of protein phosphatase homologs in animal cells that are predicted to localize to mitochondria based on sequence analysis. Interestingly, we found that Aup1p localizes to the mitochondrial intermembrane space and is required for efficient mitophagy in stationary phase cells. Viability studies demonstrate that Aup1p is required for efficient survival of cells in prolonged stationary phase cultures, implying a pro-survival role for mitophagy under our working conditions. Our data suggest that Aup1p may be part of a signal transduction mechanism that marks mitochondria for sequestration into autophagosomes.