Predictors of unexplained early neurological deterioration after thrombectomy for posterior circulation infarction: a reanalysis of the BASILAR study.

OBJECTIVE: The efficacy of endovascular thrombectomy in patients with posterior circulation ischemic stroke remains controversial. Early neurological deterioration (END) as an important predictor of poor outcome is poorly understood, except in cases of symptomatic intracranial hemorrhage, recanalization failure, and malignant cerebral edema. The objective of this study was to assess predictors of unexplained END (UnEND) after endovascular thrombectomy. METHODS: The BASILAR study is a multicenter prospective observational study in which 647 patients with vertebrobasilar occlusion on imaging within 24 hours of stroke onset and who underwent endovascular treatment were enrolled, of whom 477 who had undergone successful recanalization were included in this study. Multivariate analysis was used to identify the predictors of UnEND, defined as a ≥ 4-point increase in National Institutes of Health Stroke Scale (NIHSS) score at 24 hours after endovascular thrombectomy. RESULTS: Among the 477 eligible patients included, UnEND occurred in 86 (18%) patients. The predictors of UnEND were stress hyperglycemic ratio (SHR) (OR 2.2, 95% CI 1.1-4.6; p = 0.031), baseline NIHSS score (OR 0.9, 95% CI 0.83-0.95; p = 0.001), and asymptomatic intracerebral hemorrhage (aICH) (OR 5.9, 95% CI 1.7-20.0; p = 0.004). The occurrence rate of a favorable outcome, defined as a modified Rankin Scale score of 0-3 at 90 days, was lower in the UnEND group (5.8% vs 47.6%, p < 0.001) compared with the group without END, and the UnEND group had higher mortality at 90 days (66.3% vs 27.4%, p < 0.001). CONCLUSIONS: UnEND may be associated with poor outcome after endovascular thrombectomy in patients with acute vertebrobasilar occlusion. Some modifiable factors such as SHR and aICH could be targeted to improve the efficacy of endovascular thrombectomy.

In-depth discovery and taste presentation mechanism studies on umami peptides derived from fermented sea bass based on peptidomics and machine learning.

Umami peptides originating from fermented sea bass impart a distinctive flavor to food. Nevertheless, large-scale and rapid screening for umami peptides using conventional techniques is challenging because of problems such as prolonged duration and complicated operation. Therefore, we aimed to screen fermented sea bass using peptidomics and machine learning approaches. The taste presentation mechanism of umami peptides was assessed by molecular docking of T1R1/T1R3. Seventy umami peptides identified in fermented sea bass predominantly originated from 28 precursor proteins, including troponin, myosin, motor protein, and creatine kinase. Six umami peptides with the lowest energies formed stable complexes by binding to T1R3. SER170, SER147, GLN389, and HIS145 are critical binding sites for T1R1/T1R3. Four dominant interacting surface forces were identified: aromatic interactions, hydrogen bonding, hydrophilic bonds, and solvent-accessible surfaces. Our study unveils a method to screen umami peptides efficiently, providing a basis for further exploration of their flavor in fermented sea bass.

Two mutations at KRT74 and EDAR synergistically drive the fine-wool production in Chinese sheep.

INTRODUCTION: Fine-wool sheep are the most common breed used by the wool industry worldwide. Fine-wool sheep have over a three-fold higher follicle density and a 50% smaller fiber diameter than coarse-wool sheep. OBJECTIVES: This study aims to clarify the underlying genetic basis for the denser and finer wool phenotype in fine-wool breeds. METHOD: Whole-genome sequences of 140 samples, Ovine HD630K SNP array data of 385 samples, including fine, semi-fine, and coarse wool sheep, as well as skin transcriptomes of nine samples were integrated for genomic selection signature analysis. RESULTS: Two loci at keratin 74 (KRT74) and ectodysplasin receptor (EDAR) were revealed. Fine-scale analysis in 250 fine/semi-fine and 198 coarse wool sheep narrowed this association to one C/A missense variant of KRT74 (OAR3:133,486,008, P = 1.02E-67) and one T/C SNP in the regulatory region upstream of EDAR (OAR3:61,927,840, P = 2.50E-43). Cellular over-expression and ovine skin section staining assays confirmed that C-KRT74 activated the KRT74 protein and specifically enlarged cell size at the Huxley's layer of the inner root sheath (P < 0.01). This structure enhancement shapes the growing hair shaft into the finer wool than the wild type. Luciferase assays validated that the C-to-T mutation upregulated EDAR mRNA expression via a newly created SOX2 binding site and potentially led to the formation of more hair placodes. CONCLUSIONS: Two functional mutations driving finer and denser wool production were characterized and offered new targets for genetic breeding during wool sheep selection. This study not only provides a theoretical basis for future selection of fine wool sheep breeds but also contributes to improving the value of wool commodities.

TNIP1 inhibits selective autophagy via bipartite interaction with LC3/GABARAP and TAX1BP1.

Mitophagy is a form of selective autophagy that disposes of superfluous and potentially damage-inducing organelles in a tightly controlled manner. While the machinery involved in mitophagy induction is well known, the regulation of the components is less clear. Here, we demonstrate that TNIP1 knockout in HeLa cells accelerates mitophagy rates and that ectopic TNIP1 negatively regulates the rate of mitophagy. These functions of TNIP1 depend on an evolutionarily conserved LIR motif as well as an AHD3 domain, which are required for binding to the LC3/GABARAP family of proteins and the autophagy receptor TAX1BP1, respectively. We further show that phosphorylation appears to regulate its association with the ULK1 complex member FIP200, allowing TNIP1 to compete with autophagy receptors, which provides a molecular rationale for its inhibitory function during mitophagy. Taken together, our findings describe TNIP1 as a negative regulator of mitophagy that acts at the early steps of autophagosome biogenesis.

Effect of Controlling Light on Cashmere Growth and Harmful Gas Parameters in Shanbei White Cashmere Goats.

The quality and yield of cashmere closely affect the economic benefits of cashmere goat farming. Studies have shown that controlling light can have an important impact on cashmere but can also affect the concentration of harmful gases. In order to explore the impact of a short photoperiod on the growth of cashmere and harmful gases in goat houses, 130 female (non-pregnant) Shanbei white cashmere goats, aged 4-5 years with similar body weights, were randomly divided into a control group and a treatment group, with 65 goats in each group. The dietary nutrition levels of the experimental goats were the same, and completely natural light was used in the control group; the light control group received light for 7 h every day (9:30-16:30), and the rest of the time (16:30-9:30 the next day) they did not receive light. The light control treatment was carried out in a control house, and the gas content was analyzed. It was found that a shortened period of light exposure could increase the annual average cashmere production by 34.5%. The content of each gas has a certain functional relationship with the measurement time period, but at the same time, we found that the content of NH3 also changes seasonally. In summary, the use of shortened light periods when raising cashmere goats can significantly increase cashmere production and quality, but at the same time, it will increase the concentration of harmful gases in the goat barn, and ventilation should be increased to ensure the health of the goats and the air quality in the barn.

The implication of long non-coding RNA expression profile in rheumatoid arthritis: Correlation with treatment response to tumor necrosis factor inhibitor.

OBJECTIVE: This study aimed to investigate the linkage of long non-coding RNA (lncRNA) expression profile with etanercept response in rheumatoid arthritis (RA) patients. METHODS: Peripheral blood mononuclear cell (PBMC) samples were collected from 80 RA patients prior to etanercept treatment. Samples from eight responders and eight non-responders at week 24 (W24) were proposed to RNA-sequencing, then 10 candidate lncRNAs were sorted and their PBMC expressions were validated by reverse transcription quantitative chain reaction (RT-qPCR) in 80 RA patients. Subsequently, clinical response by lncRNA (CRLnc) prediction model was established. RESULTS: RNA-sequencing identified 254 up-regulated and 265 down-regulated lncRNAs in W24 responders compared with non-responders, which were enriched in immune or joint related pathways such as B-cell receptor signaling, osteoclast differentiation and T-cell receptor signaling pathways, etc. By reverse transcription quantitative chain reaction (RT-qPCR) validation: Two lncRNAs were correlated with W4 response, three lncRNAs were correlated with W12 response, seven lncRNAs were correlated with W24 response. Subsequently, to construct and validate CRLnc prediction model, 80 RA patients were randomly divided into test set (n = 40) and validation set (n = 40). In the test set, lncRNA RP3-466P17.2 (OR = 9.743, P = .028), RP11-20D14.6 (OR = 10.935, P = .007), RP11-844P9.2 (OR = 0.075, P = .022), and TAS2R64P (OR = 0.044, P = .016) independently related to W24 etanercept response; then CRLnc prediction model integrating these four lncRNAs presented a good value in predicting W24 etanercept response (Area Under Curve (AUC): 0.956, 95%CI: 0.896-1.000). However, in the validation set, the CRLnc prediction model only exhibited a certain value in predicting W24 etanercept response (AUC: 0.753, 95%CI: 0.536-0.969). CONCLUSIONS: CRLnc prediction model is potentially a useful tool to instruct etanercept treatment in RA patients.

Ultrasound-guided totally implantable venous access ports placement via right brachiocephalic vein in pediatric population: A clinical debut.

BACKGROUND AND OBJECTIVES: To investigate the feasibility and safety of ultrasound-guided totally implantable venous access ports (TIVAPs) via the right brachiocephalic vein (BCV) in pediatric patients. METHODS: A single-institute retrospective review was performed on 35 pediatric patients with predominantly hematological malignancies (88.6%) who underwent TIVAP implantation via ultrasound-guided right BCV approach from July 2018 to June 2021. The catheter tip was adjusted to be positioned at the cavoatrial junction under pulsed fluoroscopic guidance. Technical success rate, procedural information, and TIVAP-related complications were evaluated. RESULTS: All the pediatric TIVAP devices were successfully implanted via right BCV access. Venous access was successful by first attempt in 32 children (91%), two cases (5.7%) required a second attempt, and one patient (2.9%) required a third attempt. The mean procedural time was 44.6 ± 6.4 minutes (range: 34-62 minutes). No intraoperative complications occurred. The average TIVAP indwelling time was 564 ± 208 days (range: 193-1014 days), with a cumulative 19,723 catheter-days. Overall, three patients (8.6%) experienced four postoperative complications (two cases of local hematoma and two catheter dysfunctions) at a rate of 0.2 per 1000 catheter-days. No other complications such as wound dehiscence, delayed incision healing, catheter-related thrombosis (CRT), catheter malposition/fracture, surgical site infection, catheter-related bloodstream infection (CRBSI), pinch-off syndrome, and drug extravasation were observed during follow-up. CONCLUSIONS: Ultrasound-guided right BCV access for TIVAP placement in pediatric patients appears to be technically feasible, safe, and effective. Further large-sample, prospective studies are warranted.

The Development of Novel N-Terminal Pro-Peptide of Type I Chemiluminescence Assay.

BACKGROUND: P1NP can be used for monitoring patients treated with both bisphosphonates and teriparatide as bone formation markers. P1NP assays include two types, intact trimeric form of P1NP assay and total P1NP assay. In this study we provided another type of P1NP assay. METHODS: The α-1 chain was constructed as recombined P1NP protein in the Corynebacterium glutamicum gene expression system. Native proteins were purified from Hydrothorax. Antibody clones were screened using mice immune to the α-1 chain peptide. The screened antibody was used for assay development. Assay performance was verified and afterwards the method comparison was analyzed between the self-developed assay and Roche P1NP assay. RESULTS: α-1 chain and native P1NP proteins were purified and used for antibody selection and making the calibrator. Three clones of antibody were screened and 2 of them were used in the assay development. The assay performance was characterized, including the linearity, precision, and sensitivity. Method comparison was also performed between our assay and Roche P1NP assay showing a 0.98 slope. CONCLUSIONS: A new P1NP assay was provided that recognizes only the α-1 chain and, thus, may provide more insight for disease monitoring when the P1NP assay is applied in clinic in the future.

Preparation and Size Control of Efficient and Safe Nanopesticides by Anodic Aluminum Oxide Templates-Assisted Method.

Efficient and safe nanopesticides play an important role in pest control due to enhancing target efficiency and reducing undesirable side effects, which has become a hot spot in pesticide formulation research. However, the preparation methods of nanopesticides are facing critical challenges including low productivity, uneven particle size and batch differences. Here, we successfully developed a novel, versatile and tunable strategy for preparing buprofezin nanoparticles with tunable size via anodic aluminum oxide (AAO) template-assisted method, which exhibited better reproducibility and homogeneity comparing with the traditional method. The storage stability of nanoparticles at different temperatures was evaluated, and the release properties were also determined to evaluate the performance of nanoparticles. Moreover, the present method is further demonstrated to be easily applicable for insoluble drugs and be extended for the study of the physicochemical properties of drug particles with different sizes.

Enhancing the developmental competence of prepubertal lamb oocytes by supplementing the in vitro maturation medium with sericin and the fibroblast growth factor 2 - leukemia inhibitory factor - Insulin-like growth factor 1 combination.

Poor development of oocytes from prepubertal animals is a major factor that hinders the application of the technology, juvenile in vitro embryo transfer (JIVET). The aim of this study was to explore the possibility of improving the developmental competence of prepubertal oocytes by supplementing the oocyte in vitro maturation (IVM) medium with antioxidants and cytokines. Effects of two antioxidants, melatonin and sericin, were first examined. The results showed that melatonin had no significant beneficial roles on the lamb oocyte development, while 0.5% sericin supplemented during IVM significantly increased the blastocyst rate of lamb oocytes (46.5% vs 19.2% in control, P < 0.05). Next, effects of two kinds of combined supplements, insulin-transferrin-selenium (ITS) and fibroblast growth factor 2(FGF2)-leukemia inhibitory factor (LIF)-insulin-like growth factor1 (IGF1)(FLI) were tested. The results indicated that addition of FLI, but not ITS, in the IVM medium, significantly improved the blastocyst development of lamb oocytes (43.9% in FLI group vs 21.6% in control, P < 0.05). Further comparison showed that the developmental competence of oocytes was not significantly different among supplementation with sericin or FLI alone or both, all of which generated similar outcomes of blastocyst yield to the supplementation with adult follicular fluid. Finally, 27 blastocysts produced from lamb oocytes matured in the presence of sericin and FLI were transferred into 18 recipients, of which 9 were pregnant. This study suggests that the developmental competence of prepubertal oocytes can be improved by supplementing IVM medium with relevant agents like sericin and cytokines.

Molecular and topological reorganizations in mitochondrial architecture interplay during Bax-mediated steps of apoptosis.

During apoptosis, Bcl-2 proteins such as Bax and Bak mediate the release of pro-apoptotic proteins from the mitochondria by clustering on the outer mitochondrial membrane and thereby permeabilizing it. However, it remains unclear how outer membrane openings form. Here, we combined different correlative microscopy and electron cryo-tomography approaches to visualize the effects of Bax activity on mitochondria in human cells. Our data show that Bax clusters localize near outer membrane ruptures of highly variable size. Bax clusters contain structural elements suggesting a higher order organization of their components. Furthermore, unfolding of inner membrane cristae is coupled to changes in the supramolecular assembly of ATP synthases, particularly pronounced at membrane segments exposed to the cytosol by ruptures. Based on our results, we propose a comprehensive model in which molecular reorganizations of the inner membrane and sequestration of outer membrane components into Bax clusters interplay in the formation of outer membrane ruptures. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Association of Intake Folate and Related Gene Polymorphisms with Breast Cancer.

Breast cancer is one of the most common malignancies in women worldwide and is associated with a variety of risk factors. Folate and vitamin B12 are key elements of the one-carbon metabolism pathway where methylenetetrahydrofolate reductase (MTHFR) plays a significant role. Though many molecular and epidemiological studies have been performed to explore the relationship between intake folate, vitamin B12, MTHFR gene polymorphism and breast cancer risk, there is no consensus to date. By reviewing the relevant literatures and summarizing the potential effect of dietary folate intake on MTHFR genes polymorphism and breast cancer risk, we conclude that MTHFR C677T gene polymorphism is associated with breast cancer risk among Asian, but not Caucasians, and the MTHFR A1298C gene polymorphism is not a susceptibility factor of breast cancers. Concomitant low activity of MTHFR enzyme resulted from C677T gene polymorphism and low dietary folate intake is associated with increased breast cancer risk.

PTEN-L is a novel protein phosphatase for ubiquitin dephosphorylation to inhibit PINK1-Parkin-mediated mitophagy.

Mitophagy is an important type of selective autophagy for specific elimination of damaged mitochondria. PTEN-induced putative kinase protein 1 (PINK1)-catalyzed phosphorylation of ubiquitin (Ub) plays a critical role in the onset of PINK1-Parkin-mediated mitophagy. Phosphatase and tensin homolog (PTEN)-long (PTEN-L) is a newly identified isoform of PTEN, with addition of 173 amino acids to its N-terminus. Here we report that PTEN-L is a novel negative regulator of mitophagy via its protein phosphatase activity against phosphorylated ubiquitin. We found that PTEN-L localizes at the outer mitochondrial membrane (OMM) and overexpression of PTEN-L inhibits, whereas deletion of PTEN-L promotes, mitophagy induced by various mitochondria-damaging agents. Mechanistically, PTEN-L is capable of effectively preventing Parkin mitochondrial translocation, reducing Parkin phosphorylation, maintaining its closed inactive conformation, and inhibiting its E3 ligase activity. More importantly, PTEN-L reduces the level of phosphorylated ubiquitin (pSer65-Ub) in vivo, and in vitro phosphatase assay confirms that PTEN-L dephosphorylates pSer65-Ub via its protein phosphatase activity, independently of its lipid phosphatase function. Taken together, our findings demonstrate a novel function of PTEN-L as a protein phosphatase for ubiquitin, which counteracts PINK1-mediated ubiquitin phosphorylation leading to blockage of the feedforward mechanisms in mitophagy induction and eventual suppression of mitophagy. Thus, understanding this novel function of PTEN-L provides a key missing piece in the molecular puzzle controlling mitophagy, a critical process in many important human diseases including neurodegenerative disorders such as Parkinson's disease.

MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5.

The kinase PINK1 and ubiquitin ligase Parkin can regulate the selective elimination of damaged mitochondria through autophagy (mitophagy). Because of the demand on lysosomal function by mitophagy, we investigated a role for the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in this process. We show that during mitophagy TFEB translocates to the nucleus and displays transcriptional activity in a PINK1- and Parkin-dependent manner. MITF and TFE3, homologues of TFEB belonging to the same microphthalmia/transcription factor E (MiT/TFE) family, are similarly regulated during mitophagy. Unlike TFEB translocation after starvation-induced mammalian target of rapamycin complex 1 inhibition, Parkin-mediated TFEB relocalization required Atg9A and Atg5 activity. However, constitutively active Rag guanosine triphosphatases prevented TFEB translocation during mitophagy, suggesting cross talk between these two MiT/TFE activation pathways. Analysis of clustered regularly interspaced short palindromic repeats-generated TFEB/MITF/TFE3/TFEC single, double, and triple knockout cell lines revealed that these proteins partly facilitate Parkin-mediated mitochondrial clearance. These results illuminate a pathway leading to MiT/TFE transcription factor activation, distinct from starvation-induced autophagy, which occurs during mitophagy.

Chemogenomic profiling of endogenous PARK2 expression using a genome-edited coincidence reporter.

Parkin, an E3 ubiquitin ligase, is a central mediator of mitochondrial quality control and is linked to familial forms of Parkinson's disease (PD). Removal of dysfunctional mitochondria from the cell by Parkin is thought to be neuroprotective, and pharmacologically increasing Parkin levels may be a novel therapeutic approach. We used genome-editing to integrate a coincidence reporter into the PARK2 gene locus of a neuroblastoma-derived cell line and developed a quantitative high-throughput screening (qHTS) assay capable of accurately detecting subtle compound-mediated increases in endogenous PARK2 expression. Interrogation of a chemogenomic library revealed diverse chemical classes that up-regulate the PARK2 transcript, including epigenetic agents, drugs controlling cholesterol biosynthesis, and JNK inhibitors. Use of the coincidence reporter eliminated wasted time pursuing reporter-biased false positives accounting for ∼2/3 of the actives and, coupled with titration-based screening, greatly improves the efficiency of compound selection. This approach represents a strategy to revitalize reporter-gene assays for drug discovery.

Cytoplasmic irradiation results in mitochondrial dysfunction and DRP1-dependent mitochondrial fission.

Direct DNA damage is often considered the primary cause of cancer in patients exposed to ionizing radiation or environmental carcinogens. Although mitochondria are known to play an important role in radiation-induced cellular response, the mechanisms by which cytoplasmic stimuli modulate mitochondrial dynamics and functions are largely unknown. In the present study, we examined changes in mitochondrial dynamics and functions triggered by α particle damage to the mitochondria in human small airway epithelial cells, using a precision microbeam irradiator with a beam width of 1 µm. Targeted cytoplasmic irradiation using this device resulted in mitochondrial fragmentation and a reduction of cytochrome c oxidase and succinate dehydrogenase activity, when compared with nonirradiated controls, suggesting a reduction in respiratory chain function. In addition, mitochondrial fragmentation or fission was associated with increased expression of the dynamin-like protein DRP1, which promotes mitochondrial fission. DRP1 inhibition by the drug mdivi-1 prevented radiation-induced mitochondrial fission, but respiratory chain function in mitochondria inhibited by radiation persisted for 12 hours. Irradiated cells also showed an increase in mitochondria-derived superoxide that could be quenched by dimethyl sulfoxide. Taken together, our results provide a mechanistic explanation for the extranuclear, nontargeted effects of ionizing radiation.

Comparison of three different surgical approaches for treatment of thoracolumbar burst fracture.

OBJECTIVE: The main treatment method used for thoracolumbar fractures is open reduction and internal fixation. Commonly there are three surgical approaches: anterior, posterior and paraspinal. We attempt to compare the three approaches based on our clinical data analysis. METHODS: A group of 94 patients with Denis type A or B thoracolumbar burst fracture between March 2008 and September 2010 were recruited in this study. These patients were treated by anterior-, posterior- or paraspinal-approach reduction with or without decompression. The fracture was fixed with titanium mesh and Z-plate via anterior approach (24 patients), screw and rod system via posterior approach (38 patients) or paraspinal approach (32 patients). Clinical evaluations included operation duration, blood loss, incision length, preoperative and postoperative Oswestry disability index (ODI). RESULTS: The average operation duration (94.1 min +/- 13.7 min), blood loss (86.7 ml +/-0.0 ml), length of incision (9.3 mm +/- 0.7 mm) and postoperative ODI (6 +/- 0.5) were significantly lower (P less than 0.05) in paraspinal approach group than in traditional posterior approach group (operation duration 94.1 min +/- 13.7 min, blood loss 143.3 ml +/-28.3 ml, length of incision 15.4 cm +/- 2.1 cm and ODI 12 +/- 0.7) and anterior approach group (operation duration 176.3 min +/- 20.7 min, blood loss 255.1 ml +/- 38.4 ml, length of incision 18.6 cm +/- 2.4 cm and ODI 13 +/- 2.4). There was not statistical difference in terms of Cobb angle on radiographs among the three approaches. CONCLUSION: The anterior approach surgery is convenient for resection of the vertebrae and reconstruction of vertebral height, but it is more complicated and traumatic. Hence it is mostly used for severe Denis type B fracture. The posterior approach is commonly applied to most thoracolumbar fractures and has fewer complications compared with the anterior approach, but it has some shortcomings as well. The paraspinal approach has great advantages compared with the other two approaches. It is in accordance with the concept of minimally invasive surgery and can replace most posterior approach operations.

Effects of cytokines and chemokines on migration of mesenchymal stem cells following spinal cord injury.

We investigated the effects of cytokines and chemokines and their associated signaling pathways on mesenchymal stem cell migration after spinal cord injury, to determine their roles in the curative effects of mesenchymal stem cells. This study reviewed the effects of tumor necrosis factor-α, vascular endothelial growth factor, hepatocyte growth factor, platelet-derived growth factor, basic fibroblast growth factor, insulin like growth factor-1, stromal cell-derived factor and monocyte chemoattractant protein-1, 3 during mesenchymal stem cell migration to damaged sites, and analyzed the signal transduction pathways involved in their effects on mesenchymal stem cell migration. The results confirmed that phosphatidylinositol 3-kinase/serine/threonine protein kinases and nuclear factor-κB play crucial roles in the migration of mesenchymal stem cells induced by cytokines and chemokines.

Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol.

The Bcl-2 family member Bax translocates from the cytosol to mitochondria, where it oligomerizes and permeabilizes the mitochondrial outer membrane to promote apoptosis. Bax activity is counteracted by prosurvival Bcl-2 proteins, but how they inhibit Bax remains controversial because they neither colocalize nor form stable complexes with Bax. We constrained Bax in its native cytosolic conformation within cells using intramolecular disulfide tethers. Bax tethers disrupt interaction with Bcl-x(L) in detergents and cell-free MOMP activity but unexpectedly induce Bax accumulation on mitochondria. Fluorescence loss in photobleaching (FLIP) reveals constant retrotranslocation of WT Bax, but not tethered Bax, from the mitochondria into the cytoplasm of healthy cells. Bax retrotranslocation depends on prosurvival Bcl-2 family proteins, and inhibition of retrotranslocation correlates with Bax accumulation on the mitochondria. We propose that Bcl-x(L) inhibits and maintains Bax in the cytosol by constant retrotranslocation of mitochondrial Bax.