Assessment of sedation by automated pupillometry in critically ill patients: a prospective observational study.

BACKGROUND: Quantitative measurement of pupil change has not been assessed against the Richmond Agitation and Sedation Scale (RASS) and spectral edge frequency (SEF) during sedation. The aim of this study was to evaluate pupillometry against these measures in sedated critically ill adult patients. METHODS: In ventilated and sedated patients, pupillary variables were measured by automated pupillometry at each RASS level from -5 to 0 after discontinuation of hypnotics, while processed electroencephalogram variables were displayed continuously and SEF was recorded at each RASS level. Correlations were made between percentage pupillary light reflex (%PLR) and RASS, and between %PLR and SEF. The ability of %PLR to differentiate light sedation (RASS ≥-2), moderate (RASS =-3), and deep sedation (RASS ≤-4) was assessed by areas under receiver operating characteristic (ROC) curves. RESULTS: A total of 163 paired measurements were recorded in 38 patients. With decreasing sedation depth, median %PLR increased progressively from 20% (interquartile range 17-25%) to 36% (interquartile range 33-40%) (P<0.001). Strong correlations were found between %PLR and RASS (Rho=0.635) and between %PLR and SEF (R=0.641). Area under the curve (AUC) of 0.87 with a %PLR threshold of 28% differentiated moderate/light sedation from deep sedation with sensitivity of 83% and specificity of 83%. An AUC of 0.82 with a threshold of 31% distinguished light sedation from moderate/deep sedation with a sensitivity of 81% and a specificity of 75%. CONCLUSIONS: Quantitative assessment of %PLR correlates with other indicators of sedation depth in critically ill patients.

Crizotinib-based proteolysis targeting chimera suppresses gastric cancer by promoting MET degradation.

As one of the common malignant cancer types, gastric cancer (GC) is known for late-stage diagnosis and poor prognosis. Overexpression of the receptor tyrosine kinase MET is associated with poor prognosis among patients with advanced stage GC. However, no MET inhibitor has been used for GC treatment. Like other tyrosine kinase inhibitors that fit the "occupancy-driven" model, current MET inhibitors are prone to acquired resistance. The emerging proteolysis targeting chimera (PROTAC) strategy could overcome such limitations through direct degradation of the target proteins. In this study, we successfully transformed the MET-targeted inhibitor crizotinib into a series of PROTACs, recruiting cereblon/cullin 4A E3 ubiquitin ligase to degrade the MET proteins. The optimized lead PROTAC (PRO-6 E) effectively eliminated MET proteins in vitro and in vivo, inhibiting proliferation and motility of MET-positive GC cells. In the MKN-45 xenograft model, PRO-6 E showed pronounced antitumor efficacy with a well-tolerated dosage regimen. These results validated PRO-6 E as the first oral PROTAC for MET-dependent GC.

Tapered-cuff Endotracheal Tube Does Not Prevent Early Postoperative Pneumonia Compared with Spherical-cuff Endotracheal Tube after Major Vascular Surgery: A Randomized Controlled Trial.

BACKGROUND: Patients undergoing major vascular surgery often develop postoperative pneumonia that impacts their outcomes. Conflicting data exist concerning the potential benefit of tapered-shaped cuffs on tracheal sealing. The primary objective of this study was to assess the efficiency of a polyvinyl chloride tapered-cuff endotracheal tube at reducing the postoperative pneumonia rate after major vascular surgery. Secondary objectives were to determine its impact on microaspiration, ventilator-associated pneumonia rate, and inner cuff pressure. METHODS: This prospective randomized controlled study included 109 patients who were randomly assigned to receive either spherical- (standard cuff) or taper-shaped (tapered cuff) endotracheal tubes inserted after anesthesia induction and then admitted to the intensive care unit after major vascular surgery. Cuff pressure was continuously recorded over 5 h. Pepsin and α-amylase concentrations in tracheal aspirates were quantified on postoperative days 1 and 2. The primary outcome was the early postoperative pneumonia frequency. RESULTS: Comparing the tapered-cuff with standard-cuff group, respectively, postoperative pneumonia rates were comparable (42 vs. 44%, P = 0.87) and the percentage (interquartile range) of cuff-pressure time with overinflation was significantly higher (16.1% [1.5 to 50] vs. 0.6% [0 to 8.3], P = 0.01), with a 2.5-fold higher coefficient of variation (20.2 [10.6 to 29.4] vs. 7.6 [6.2 to 10.2], P < 0.001). Although microaspiration frequencies were high, they did not differ between groups. CONCLUSION: For major vascular surgery patients, polyvinyl chloride tapered-cuff endotracheal tubes with intermittent cuff-pressure control did not lower the early postoperative pneumonia frequency and did not prevent microaspiration.

Early fluid loading in acute respiratory distress syndrome with septic shock deteriorates lung aeration without impairing arterial oxygenation: a lung ultrasound observational study.

INTRODUCTION: The study was designed to assess the impact of fluid loading on lung aeration, oxygenation and hemodynamics in patients with septic shock and acute respiratory distress syndrome (ARDS). METHODS: During a 1-year period, a prospective observational study was performed in 32 patients with septic shock and ARDS. Cardiorespiratory parameters were measured using Swan Ganz (n = 29) or PiCCO catheters (n = 3). Lung aeration and regional pulmonary blood flows were measured using bedside transthoracic ultrasound. Measurements were performed before (T0), at the end of volume expansion (T1) and 40 minutes later (T2), consisting of 1-L of saline over 30 minutes during the first 48 h following onset of septic shock and ARDS. RESULTS: Lung ultrasound score increased by 23% at T2, from 13 at baseline to 16 (P < 0.001). Cardiac index and cardiac filling pressures increased significantly at T1 (P < 0.001) and returned to control values at T2. The increase in lung ultrasound score was statistically correlated with fluid loading-induced increase in cardiac index and was not associated with increase in pulmonary shunt or regional pulmonary blood flow. At T1, PaO2/FiO2 significantly increased (P < 0.005) from 144 (123 to 198) to 165 (128 to 226) and returned to control values at T2, whereas lung ultrasound score continued to increase. CONCLUSIONS: Early fluid loading transitorily improves hemodynamics and oxygenation and worsens lung aeration. Aeration changes can be detected at the bedside by transthoracic lung ultrasound, which may serve as a safeguard against excessive fluid loading.

Unusual 5/5 fused bicyclosesquiterpenoids from Eupatorium adenophorum.

The chemical analysis on the aerial sections of Eupatorium adenophorum Spreng. resulted in the identification of four unprecedented 5/5 fused bicyclosesquiterpenoids, eupatorid A (1), and its analogues named eupatorester A-C (2-4) using various chromatographic techniques. Their structures were unambiguously confirmed by detailed spectroscopic investigations (including 1D, 2D-NMR and HRMS), and single crystal X-ray diffraction. The anti-inflammatory activities, in vitro tumor growth inhibitory activities and antibacterial activities of these compounds were evaluated.

FXR agonist GW4064 enhances anti-PD-L1 immunotherapy in colorectal cancer.

Colorectal cancer (CRC) is one of the top three malignant tumors in terms of morbidity, and the limited efficacy of existing therapies urges the discovery of potential treatment strategies. Immunotherapy gradually becomes a promising cancer treatment method in recent decades; however, less than 10% of CRC patients could really benefit from immunotherapy. It is pressing to explore the potential combination therapy to improve the immunotherapy efficacy in CRC patients. It is reported that Farnesoid X receptor (FXR) is deficiency in CRC and associated with immunity. Herein, we found that GW4064, a FXR agonist, could induce apoptosis, block cell cycle, and mediate immunogenic cell death (ICD) of CRC cells in vitro. Disappointingly, GW4064 could not suppress the growth of CRC tumors in vivo. Further studies revealed that GW4064 upregulated PD-L1 expression in CRC cells via activating FXR and MAPK signaling pathways. Gratifyingly, the combination of PD-L1 antibody with GW4064 exhibited excellent anti-tumor effects in CT26 xenograft models and increased CD8+ T cells infiltration, with 33% tumor bearing mice cured. This paper illustrates the potential mechanisms of GW4064 to upregulate PD-L1 expression in CRC cells and provides important data to support the combination therapy of PD-L1 immune checkpoint blockade with FXR agonist for CRC patients.

Three new cadinene-type sesquiterpenoids from the aerial parts of Ageratina adenophora.

Three new cadinene sesquiterpenoids 1-3, were isolated from the aerial sections of Ageratina adenophora using various chromatographic techniques. Their structures were characterised by comprehensive spectroscopic investigations (including 1D, 2D-NMR and HRMS), and single crystal X-ray diffraction. The cytotoxic activity of new compounds 1-3 were evaluated by testing in vitro tumour growth inhibitory rate against five human tumour cell lines, HL-60, A-549, SMMC-7721, MDA-MB-231, and SW480.

Innate Root Exudates Contributed to Contrasting Coping Strategies in Response to Ralstonia solanacearum in Resistant and Susceptible Tomato Cultivars.

Tomato cultivars with contrasting resistance to pathogens regulate root exudates differentially in response to Ralstonia solanacearum attacks. However, strategies using innate root exudates against infection remain unknown. This study analyzed the innate root exudates of two tomato cultivars and their functions in regulating R. solanacearum infection. The innate root exudates differed between the two cultivars. Astaxanthin released from resistant plants inhibited colonization by R. solanacearum but promoted motility, while neferine released from susceptible plants suppressed motility and colonization. The secretion of astaxanthin in resistant tomatoes promoted the growth of biocontrol fungi in soil and reduced the abundance of pathogenic fungi. Neferine secreted by the susceptible cultivar inhibited the relative abundance of the bacterial-biocontrol-related Bacillus genus, indirectly reducing the soil's immune capacity. This study revealed contrasting strategies using root exudates in resistant and susceptible tomato cultivars to cope with R. solanacearum infection, providing a basis for breeding disease-resistant cultivars.

Effects of positive end-expiratory pressure titration and recruitment maneuver on lung inflammation and hyperinflation in experimental acid aspiration-induced lung injury.

BACKGROUND: In acute lung injury positive end-expiratory pressure (PEEP) and recruitment maneuver are proposed to optimize arterial oxygenation. The aim of the study was to evaluate the impact of such a strategy on lung histological inflammation and hyperinflation in pigs with acid aspiration-induced lung injury. METHODS: Forty-seven pigs were randomly allocated in seven groups: (1) controls spontaneously breathing; (2) without lung injury, PEEP 5 cm H2O; (3) without lung injury, PEEP titration; (4) without lung injury, PEEP titration + recruitment maneuver; (5) with lung injury, PEEP 5 cm H2O; (6) with lung injury, PEEP titration; and (7) with lung injury, PEEP titration + recruitment maneuver. Acute lung injury was induced by intratracheal instillation of hydrochloric acid. PEEP titration was performed by incremental and decremental PEEP from 5 to 20 cm H2O for optimizing arterial oxygenation. Three recruitment maneuvers (pressure of 40 cm H2O maintained for 20 s) were applied to the assigned groups at each PEEP level. Proportion of lung inflammation, hemorrhage, edema, and alveolar wall disruption were recorded on each histological field. Mean alveolar area was measured in the aerated lung regions. RESULTS: Acid aspiration increased mean alveolar area and produced alveolar wall disruption, lung edema, alveolar hemorrhage, and lung inflammation. PEEP titration significantly improved arterial oxygenation but simultaneously increased lung inflammation in juxta-diaphragmatic lung regions. Recruitment maneuver during PEEP titration did not induce additional increase in lung inflammation and alveolar hyperinflation. CONCLUSION: In a porcine model of acid aspiration-induced lung injury, PEEP titration aimed at optimizing arterial oxygenation, substantially increased lung inflammation. Recruitment maneuvers further improved arterial oxygenation without additional effects on inflammation and hyperinflation.

The multifaceted involvement of exosomes in tumor progression: Induction and inhibition.

As key performers in intercellular communication, exosomes released by tumor cells play an important role in cancer development, including angiogenesis, cancer-associated fibroblasts activation, epithelial-mesenchymal transformation (EMT), immune escape, and pre-metastatic niche formation. Meanwhile, other cells in tumor microenvironment (TME) can secrete exosomes and facilitate tumor progression. Elucidating mechanisms regarding these processes may offer perspectives for exosome-based antitumor strategies. In this review, we mainly introduce the versatile roles of tumor or stromal cell derived exosomes in cancer development, with a particular focus on the biological capabilities and functionalities of their diverse contents, such as miRNAs, lncRNAs, and circRNAs. The potential clinical application of exosomes as biomarkers in cancer diagnosis and prognosis is also discussed. Finally, the current antitumor strategies based on exosomes in immunotherapy and targeted delivery for chemotherapeutic or biological agents are summarized.

Targeting Endothelial Cell-Specific Molecule 1 Protein in Cancer: A Promising Therapeutic Approach.

Despite the dramatic advances in cancer research in the past few years, effective therapeutic strategies are urgently needed. Endothelial cell-specific molecule 1 (ESM-1), a soluble dermatan sulfate proteoglycan, also known as endocan, serves as a diagnostic and prognostic indicator due to its aberrant expression under pathological conditions, including cancer, sepsis, kidney diseases, and cardiovascular disease. Significantly, ESM-1 can promote cancer progression and metastasis through the regulation of tumor cell proliferation, migration, invasion, and drug resistant. In addition, ESM-1 is involved in the tumor microenvironment, containing inflammation, angiogenesis, and lymph angiogenesis. This article reviews the molecular and biological characteristics of ESM-1 in cancer, the underlying mechanisms, the currently clinical and pre-clinical applications, and potential therapeutic strategies. Herein, we propose that ESM-1 is a new therapeutic target for cancer therapy.

[Preparation and application of the national reference panel for hepatitis B virus DNA].

OBJECTIVES: To calibrate the national hepatitis B virus (HBV) DNA standard according to world health organization's standard material and prepare the national reference panel for HBV DNA reagents. METHODS: Sera from blood donors and HBV patients were collected and detected by home-made HBV DNA PCR kits, HBsAg kits, and anti-HBc kits, and then confirmed by HBV DNA PCR kits produced by Roche in German, which was recognized by the world health organization. The stability of the panel was detected by acceleration method. RESULTS: The convinced copies of the sensitivity samples were gotten by seven independent experiments, the coefficients of variation of logarithm of the copies of L0-L5 were all less than 15%. Regarding the national reference panel as the standard, the quality of most domestic HBV DNA PCR kits was improved, while part of the kits should be further qualified. CONCLUSION: The national reference panel for HBV DNA reagents is developed. It contains eight negative, nine positive sera and seven samples for sensitivity test

[Effects of microbial agent inoculation on bacterial community diversity in the process of pig manure composting].

PCR-DGGE method was adopted to study the effects of inoculating exogenous microbial agent on the bacterial community diversity in the process of fresh pig manure high-temperature aerobic composting. Exogenous microbial agent inoculation promoted the composting process, with the high-temperature period being advanced by 2 days than that of non-inoculation. DGGE pattern analysis showed that during composting, the dominant bacteria changed significantly, with the Shannon-Wiener index of bacterial community at different composting periods varied obviously. The sequencing of DGGE-distinguished bands showed that Clostridium stercorarium subsp. thermolacticum sp. was the dominant group in the whole composting process. Uncultured bacteria Bacillus coagulans sp. and Clostridium thermocellum sp. became the main groups on the 10th and 16th day after microbial agent inoculation, while uncultured Firmicutes sp. and delta proteobacterium became the dominant groups on the 5th and 16th days in the treatment non-inoculation, respectively. Un-dominant group Ureibacillus thermosphaericu sp. and uncultured Silvimonas sp. appeared in the late period of well rotted composting, while uncultured soil bacteria mainly appeared in the initial and high-temperature periods. UPGMC cluster analysis showed that exogenous microbial agent inoculation obviously affected the bacterial community structure in different composting periods, and the main component analysis of DGGE patterns in composting process showed that the bacterial community was mainly affected by the exogenous microbial agent inoculation.

[Evaluation on the analytical sensitivity of 31 HBsAg enzyme immunoassay kits].

OBJECTIVE: To study the analytical sensitivity on 31 HBsAg enzyme immunoassy (EIA) test kits. METHODS: Thirty one HBsAg EIA kits produced by domestic or overseas manufactories and applied for approval during May 2007 to May 2008, were evaluated using the national reference panels. The hyperbolic curve of the log A value and log concentration for the national sensitivity standards was established. The cut-off value of each kit was substituted into the curvilinear equation to determine the analytical sensitivity which was compared between different HBsAg EIA kits. RESULTS: Twenty seven (351 lots) domestic and 4 (27 lots) overseas kits were compared. Among 378 lots of the 31 HBsAg EIA kits, only 2 lots of the domestic kits had a lower sensitivity when tested with the national HBsAg reference panels, with an average approvalr ate of 99.43% (349/351). The mean analytical sensitivity of the domestic kits for adr, adw, ay serotypes were 0.307, 0.419, 0.513 ng/ml, respectively. There was a significant difference between serotypes (F = 97.30, P < 0.01). The mean analytical sensitivity of the overseas kits for adr, adw, ay serotypes were 0.054, 0.066, 0.050 ng/ml respectively, with no significant difference between serotypes (F = 0.65, P > 0.05). The analytical sensitivity of the overseas kits for all the three serotypes was higher than that of the domestic kits (P < 0.01). There was no significant difference found between the analytical sensitivities of the kits produced by the same manufactory using 30- or 60-minute incubation of detection (P > 0.05). In contrast, there was significant difference noticed between the analytical sensitivities of the kits produced by the same manufactory when tested for 10 or 15-minute coloration of the results (P < 0.01). CONCLUSION: Analytical sensitivity of the HBsAg EIA domestic kits should be further improved, especially for detecting adw and ay serotypes.

[Preparation of the national reference panel for hepatitis B surface antigen].

OBJECTIVE: To establish the national quantity standard of hepatitis B surface antigen according to the world health organization' s standard material and prepare the national liner reference panel for hepatitis B surface antigen. METHODS: Sera from hepatitis B patients and health blood donors in different areas were collected and detected by domastic HBsAg kits, anti-HBs kits, HBeAg kits, anti-HBe kits, anti-HBc kits and anti-HCV, and then confirmed by the kits produced by Abbott, which was approved by WHO. One serum with high concentration of HBsAg was calibrated with the standard sample of WHO. And then it was diluted by 1.5 fold as the liner HBsAg reference panel. RESULTS: The HBsAg concentration of one serum was 1226 IU/ml calibrated by 21 independent standardization measurements with 7 kinds of kits. The coefficient of variation of each calibration were less then 15%. A panel contained 8 serial dilutions was established as the national liner HBsAg reference panel. The permitted range of every dilution was stipulated and the stability of the panel was detected by accelerated test. CONCLUSIONS: The national quantity standard of hepatitis B surface antigen was established and the national quantitative reference panel for HBsAg which contains eight liner serum was developed.

[Evaluation of multiplex nucleic acid testing assays for screening of hepatitis B virus DNA in blood donation process].

OBJECTIVE: To evaluate the multiplex nucleic acid testing (NAT) assays for HBV, HCV and HIV in detecting HBV DNA in plasma samples. METHODS: 534 plasma samples collected form several areas were detected with Abbott Architect i2000 HBsAg, ani-HBs, HBeAg, anti-HBe, anti-HBc and anti-HBc IgM diagnostic kits. HBV DNA levels of those samples were detected with Roche COBAS AmpliPrep/COBAS TaqMan HBV Test. Two kinds of multiplex NAT assays for HBV, HCV and HIV were used to test HBV DNA of those 534 samples. Results of serology-markers and quantitative HBV DNA levels with results of NAT were compared. RESULTS: HBV DNA was positive in all 81 HBsAg, HBeAg and anti-HBc positive samples, detected by both of NAT assays. HBV DNA was positive in 11 and 19 of 200 HBsAg negative samples when detected with the two kinds of NAT assays separately. Compared with the quantitative results detected by Roche COBAS AmpliPrep/COBAS TaqMan HBV Test, the HBV DNA positive rates were 96.9% and 94.3% in 193 samples of HBV DNA levels over 500 IU/ml while 40.2% and 45.3% in 117 samples of HBV DNA levels below 500 IU/ml while 99.3% and 96.0% in 151 samples of DNA negative HBV. CONCLUSION: There are some occult low level HBV DNA carriers with HBsAg negative results in China. NAT assays for HBV, HCV and HIV may be useful to improve the transfusion safety.

[Primary evaluation of anti-HEV diagnostic reagent by experimental infection animal model with hepatitis E virus].

OBJECTIVE: To evaluate anti-HEV diagnostic kits by experimental infecting rhesus monkeys with HEV. METHODS: Eight rhesus monkeys were infected with genotype 1 and 4 HEV separately. The alanine aminotransferase (ALT) level of all monkeys were detected before and after the process of infection. HEV RNA in stool specimens was tested by reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Anti-HEV IgG in serum was detected by GL-IgG and WT-IgG. RESULTS: HEV RNA presented in the stool of all the 8 monkeys after infection. The ALT level of 1 monkey infected with genotype 1 HEV and 2 monkeys infected with genotype 4 HEV appeared abnormally after infection. Tested by GL-IgG, 2 of the 4 monkeys infected with genotype 1 HEV and 1 of 4 monkeys infected with genotype 4 HEV seroconverted to anti-HEV IgG. However, when tested by WT-IgG, all the infected monkeys seroconverted to anti-HEV IgG. The anti-HEV IgG tested by WT-IgG was positive during the whole observation period,and the anti-HEV IgG measured by GL-IgG only remained 12 weeks after infection. Detected by GL-IgG and WT-IgG, seropositive conversion of the anti-HEV IgG happened almost at the same time. CONCLUSION: Both GL-IgG and WT-IgG could detect the anti-HEV IgG of experimentally infected rhesus monkeys but the WT-IgG had a higher sensitivity for detection of anti-HEV IgG than

[Clinical significance of detecting RNA and anti HEV antibody in convalesent sera in patients with acute HEV hepatitis].

OBJECTIVE: To investigate the anti hepatitis E virus (HEV) and HEV RNA in acute and convalescent sera of patients with NonA-E acute hepatitis. METHODS: The serum samples were taken from 95 patients who were diagnosed as acute NonA-E hepatitis. Enzyme immunoassay (EIA) was used for detecting anti-HEV Immunoglobulin G (IgG, Genolable and Wantai EIA anti-HEV kits). RT-PCR amplification of HEV RNA was based on the open reading frame 2 region of HEV and the PCR products were sequenced. RESULTS: Sera from 95 patients who were negative for anti-HEV in acute phase were followed up for 11-35 days to detect the anti-HEV antibody in recovery phase, 16/95 (16.84%) were positive for anti-HEV (wantai EIA anti-HEV kits). Ten (62.50%) were positive for HEV RNA in acute phase. Sequence analysis showed that 4 were HEV genotype. 6 were HEV genotype; 12/95 (12.50%) were positive for anti-HEV (Genolable EIA anti-HEV kits). Seven were positive for HEV RNA; 4 belonged to HEV genotype, 3 were HEV genotype. CONCLUSION: It is significant and necessary to detect anti HEV antibody and HEV RNA in patients with HEV infection during acute phase and convalesent phase.

Parkin and CASK/LIN-2 associate via a PDZ-mediated interaction and are co-localized in lipid rafts and postsynaptic densities in brain.

Mutations in the gene encoding parkin cause an autosomal recessive juvenile-onset form of Parkinson's disease. Parkin functions as a RING-type E3 ubiquitin-ligase, coordinating the transfer of ubiquitin to substrate proteins and thereby targeting them for degradation by the proteasome. We now report that the extreme C terminus of parkin, which is selectively truncated by a Parkinson's disease-causing mutation, functions as a class II PDZ-binding motif that binds CASK, the mammalian homolog of Caenorhabditis elegans Lin-2, but not other PDZ proteins in brain extracts. Importantly, parkin co-localizes with CASK at synapses in cultured cortical neurons as well as in postsynaptic densities and lipid rafts in brain. Further, parkin associates not only with CASK but also with other postsynaptic proteins in the N-methyl d-aspartate (NMDA) receptor-signaling complex, in rat brain in vivo. Finally, despite exhibiting E2-dependent ubiquitin ligase activity, rat brain parkin does not ubiquitinate CASK, suggesting that CASK may function in targeting or scaffolding parkin within the postsynaptic complex rather than as a direct substrate for parkin-mediated ubiquitination. These data implicate for the first time a PDZ-mediated interaction between parkin and CASK in neurodegeneration and possibly in ubiquitination of proteins involved in synaptic transmission and plasticity.

Parkin prevents mitochondrial swelling and cytochrome c release in mitochondria-dependent cell death.

Parkin gene mutations have been implicated in autosomal-recessive early-onset parkinsonism and lead to specific degeneration of dopaminergic neurons in midbrain. To investigate the role of Parkin in neuronal cell death, we overproduced this protein in PC12 cells in an inducible manner. In this cell line, neuronally differentiated by nerve growth factor, Parkin overproduction protected against cell death mediated by ceramide, but not by a variety of other cell death inducers (H(2)O(2), 4-hydroxynonenal, rotenone, 6-OHDA, tunicamycin, 2-mercaptoethanol and staurosporine). Protection was abrogated by the proteasome inhibitor epoxomicin and disease-causing variants, indicating that it was mediated by the E3 ubiquitin ligase activity of Parkin. Interestingly, Parkin acted by delaying mitochondrial swelling and subsequent cytochrome c release and caspase-3 activation observed in ceramide-mediated cell death. Subcellular fractionation demonstrated enrichment of Parkin in the mitochondrial fraction and its association with the outer mitochondrial membrane. Together, these results suggest that Parkin may promote the degradation of substrates localized in mitochondria and involved in the late mitochondrial phase of ceramide-mediated cell death. Loss of this function may underlie the degeneration of nigral dopaminergic neurons in patients with Parkin mutations.