Lung protection effect of EIT-based individualized protective ventilation strategy in patients with partial pulmonary resection.

OBJECTIVE: This study aimed to evaluate the lung protection effect of an individualized protective ventilation strategy based on lung impedance tomography (EIT) technology in patients with partial pulmonary resection. PATIENTS AND METHODS: Eighty patients of any gender, American Society of Anesthesiologists (ASA) classification I-II, age 30-64 years and body mass index (BMI) 18-28 kg/m2 who underwent elective thoracoscopic partial lung resection were selected and divided into 2 groups (n=40) using the random number table method: [positive end-expiratory pressure (PEEP) by electrical impedance tomography (EIT)] PEEPEIT group (experimental group) and control group. The PEEPEIT group used volume-controlled ventilation after one-lung ventilation, setting a tidal volume of 6 ml/kg and titrating the optimal PEEP value by EIT. Group C used volume-controlled ventilation after one-lung ventilation, setting a tidal volume of 6 ml/kg and a PEEP of 5 cm H2O. Clinical data were collected and recorded at 5 min after double lung ventilation (T0), single lung ventilation, 30 min after PEEP setting (T1), 60 min after PEEP setting (T2), the end of surgery, 10 min after resumption of double lung ventilation (T3) and 10 min after removal of the tracheal tube (T4), and serum surface active substance-associated protein-A (SP-A) concentrations were measured at T0, T3 and 1 d after surgery (T5). RESULTS: PEEP values were higher in the PEEPEIT group than in the control group at T1 and T2 (p-value <0.05); oxygenation index (OI) was higher in the PEEPEIT group compared to the control group at T2 and T3 (p-value <0.05); pulmonary dynamic compliance (Cdyn) was higher in the PEEPEIT group compared to the control group at T1 and T2 (p-value <0.05); intrapulmonary shunt rate (Qs/Qt) was lower in the PEEPEIT group compared to the control group at T1, T2 and at T3, the intrapulmonary shunt rate (Qs/Qt) was reduced in the PEEPEIT group compared to group C (p-value <0.05); at T5, the SP-A protein was reduced in the PEEPEIT group compared to group C. There was no statistically significant difference in the incidence of postoperative pulmonary complications between the two groups (p-value >0.05). CONCLUSIONS: The EIT-guided individualized protective ventilation strategy has a lung-protective effect in patients undergoing thoracoscopic partial lung resection.

[Clinical features and outcomes of newly diagnosed follicular lymphoma concurrent with diffuse large B-cell lymphoma component].

Objective: To explore the clinical features and survival of newly diagnosed follicular lymphoma (FL) patients with diffuse large B-cell lymphoma (DLBCL) component. Methods: 1845 newly diagnosed FL patients aged ≥ 18 years with grades 1-3a in 11 medical centers in China from 2000 to 2020 were included, and patients with DLBCL component were screened. The clinical data and survival data of the patients were retrospectively analyzed, and the prognostic factors were screened by univariate and multivariate analysis. Results: 146 patients (7.9% ) with newly diagnosed FL had DLBCL component. The median age was 56 (25-83) years, 79 males (54.1% ) . The pathology of 127 patients showed the proportion of DLBCL component. Patients were divided into two groups according to whether the proportion of DLBCL component was ≥ 50% . The study found that patients with DLBCL component ≥ 50% had higher grade 3 ratio (94.3% vs 91.9% , P=0.010) , Ki-67 index ≥ 70% ratio (58.5% vs 32.9% , P=0.013) and PET-CT SUVmax ≥ 13 ratio (72.4% vs 46.3% , P=0.030) than patients with DLBCL component<50% . All patients received CHOP or CHOP like ± rituximab chemotherapy. The overall response rate (ORR) was 88.2% , and the complete response (CR) rate was 76.4% . In the groups with different proportions of DLBCL component, there was no significant difference in the remission rate after induction treatment and the incidence of disease progression within 2 years after initiation of treatment (POD24) (P<0.05) . The overall estimated 5-year progression free survival (PFS) rate was 58.9% , and the 5-year overall survival (OS) rate was 90.4% . The 5-year OS rate of POD24 patients was lower than that of non POD24 patients (70.3% vs 98.5% , P<0.001) . Compared with non maintenance treatment of rituximab, maintenance treatment of rituximab could not benefit the 5-year PFS rate (57.7% vs 58.8% , P=0.543) , and the 5-year OS rate had a benefit trend, but the difference was not statistically significant (100% vs 87.8% , P=0.082) . Multivariate analysis showed that failure to reach CR after induction treatment was an independent risk factor for PFS (P=0.006) , while LDH higher than normal was an independent risk factor for OS (P=0.031) . Conclusion: FL patients with DLBCL component ≥50% have more invasive clinical and pathological features. CHOP/CHOP like ± rituximab regimen can improve the clinical efficacy of patients. Rituximab maintenance therapy can not benefit the PFS and OS of patients. Failure to reach CR after induction therapy was the independent unfavorable factor for PFS.

[Venetoclax with low-dose cytarabine for patients with untreated acute myeloid leukemia ineligible for intensive chemotherapy: results from the Chinese cohort of a phase three randomized placebo-controlled trial].

Objective: To investigate the safety and efficacy of venetoclax with low-dose cytarabine (LDAC) in Chinese patients with acute myeloid leukemia (AML) who are unable to tolerate intensive induction chemotherapy. Methods: Adults ≥ 18 years with newly diagnosed AML who were ineligible for intensive chemotherapy were enrolled in this international, randomized, double-blind, placebo-controlled trial. Globally, patients (n=211) were randomized 2∶1 to either venetoclax with LDAC or placebo with LDAC in 28-d cycles, with LDAC on days 1-10. The primary endpoint was OS; the secondary endpoints included response rates, event-free survival, and adverse events. Results: A total of 15 Chinese patients were enrolled (venetoclax arm, n=9; placebo arm, n=6) . The median age was 72 years (range, 61-86) . For the primary analysis, the venetoclax arm provided a 38% reduction in death risk compared with the placebo[hazard ratio (HR) , 0.62 (95%CI 0.12-3.07) ]. An unplanned analysis with an additional 6 months of follow-up demonstrated a median OS of 9.0 months for venetoclax compared with 4.1 months for placebo. The complete remission (CR) rates with CR with incomplete blood count recovery (CRi) were 3/9 (33%) and 0/6 (0%) , respectively. The most common non-hematologic adverse effects (venetoclax vs placebo) were hypokalemia[5/9 (56%) vs 4/6 (67%) ], vomiting[4/9 (44%) vs 3/6 (50%) ], constipation[2/9 (22%) vs 4/6 (67%) ], and hypoalbuminemia[1/9 (11%) vs 4/6 (67%) ]. Conclusion: Venetoclax with LDAC demonstrated meaningful efficacy and a manageable safety profile in Chinese patients consistent with the observations from the global VIALE-C population, making it an important treatment option for patients with newly diagnosed AML who are otherwise ineligible for intensive chemotherapy.

I125 irradiation stent for treatment of hepatocellular carcinoma with portal vein thrombosis: A meta-analysis.

PURPOSE: A meta-analysis aimed to systematically evaluate the safety and efficiency of I125 irradiation stent placement for patients with hepatocellular carcinoma (HCC) combined with portal vein tumor thrombosis (PVTT). MATERIALS AND METHODS: The Cochrane library, PubMed/Medline, EMBASE, CNKI, Wanfang Data and CQVIP were systematically screened out from the earliest to December 2019. The qualities of all included studies were assessed. The primary endpoints were the 6-month, 12-month stent cumulative patency rate and 6-month, 12-month, 24-month overall survival rate while the secondary endpoints were the objective response rate of PVTT, main portal venous pressure changes and treatment-related adverse events. Our meta-analysis was conducted using Stata 12.0 software. RESULTS: Totally seven studies with 1018 patients were included in the final analysis, in which 602 patients received TACE and I125 irradiation stent placement, and 416 patients in control group underwent TACE and stent placement without endovascular brachytherapy (EVBT). Meta-analysis showed that the I125 irradiation stent improved the cumulative stent patency rates in 6months [OR=1.65, 95% CI (1.32-2.05), P<0.001] and 12months [OR=2.55, 95% CI (1.90-3.42), P<0.001] and the survival rates in 6months [OR=1.77, 95% CI (1.41-2.22), P<0.001], 12months [OR=3.14, 95% CI (2.24-4.40), P<0.001] and 24months [OR=7.39, 95% CI (3.55-15.41), P<0.001]. However, there was no difference in the objective response rate of PVTT [OR=1.13, 95% CI (0.87-1.48), P=0.365], main portal venous pressure and the occurrence adverse event [OR=0.88, CI=0.72-1.08, P=0.212] between two groups. CONCLUSION: I125 irradiation stent seems to be more effective in treating hepatocellular carcinoma with portal vein tumor thrombosis. The usage of portal vein stent combined endovascular brachytherapy has the potential to act as an alternative therapy for HCC with PVTT. On account of the limitation of studies included, more studies with high-level evidence, such as RCTs, are requisite to support the above promising results.

[Clinical characteristics and ketogenic diet therapy of glucose transporter type 1 deficiency syndrome in children: a multicenter clinical study].

Objective: To explore the clinical characteristics of pediatric glucose transporter type 1 deficiency syndrome (GLUT1 DS), evaluate the efficacy and safety of ketogenic diet therapy (KDT). Methods: Clinical data of 19 children with GLUT1 DS admitted to Children's Hospital of Fudan University, Tianjin Children's Hospital, Shenzhen Children's Hospital, Children's Hospital of Nanjing Medical University and Jiangxi Provincial Children's Hospital between 2015 and 2019 were collected retrospectively. The first onset symptom, main clinical manifestations, cerebrospinal fluid features and genetic testing results of patients were summarized, the efficacy and safety of ketogenic diet treatment were analyzed. Results: Among the 19 cases, 13 were males and 6 females. The age of onset was 11.0 (1.5-45.0) months,the age of diagnosis was 54.0 (2.8-132.0) months. Epilepsy was the first onset symptom of 13 cases. Different forms of tonic-clonic seizures were the most common types of epilepsy (7 cases with generalized tonic-clonic seizures, 5 cases with focal tonic or clonic seizures, 4 cases with generalized tonic seizures). Antiepileptic drugs were effective in 4 cases. Paroxysmal motor dysfunction was present in 12 cases and ataxia was the most common one. All patients had different degrees of psychomotor retardation. Among 17 patients received cerebrospinal fluid examination, cerebrospinal fluid (CSF) glucose level was lower than 2.2 mmol/L and CSF glucose/glycemic index was<0.45 in 16 cases, only 1 case presented normal CSF glucose level (2.3 mmol/L) and normal CSF glucose/glycemic index(0.47). SLC2A1 gene mutations were found in 16 patients, missense, frameshift and nonsense mutations were the common types with 5 cases, 5 cases and 3 cases respectively. All 19 patients were treated with ketogenic diet, which was effective in 18 cases in seizure control, 11 cases in dyskinesia improvement and 18 cases in cognitive function improvement. No serious side effects were reported in any stage of KDT. Conclusions: The diagnosis of GLUT1 DS is often late. It is necessary to improve the early recognition of the disease and perform CSF glucose detection and genetic testing as early as possible. The KDT is an effective and safe treatment for GLUT1 DS, but a small number of patients have not response to diet therapy.

MiR-655-3p inhibited proliferation and migration of ovarian cancer cells by targeting RAB1A.

OBJECTIVE: To investigate the potential effect of microRNA-655-3p (miR-655-3p) on the development of ovarian cancer (OC) and its relevant mechanism. PATIENTS AND METHODS: Expression level of miR-655-3p in OC tissues was detected. The potential target gene of miR-655-3p was firstly predicted online and subsequently verified by luciferase reporter assay and Western blot. In vitro effects of miR-655-3p on SKOV3 cells were determined as well. RESULTS: Low expression of miR-655-3p in OC was confirmed by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Ras-related protein Rab-1A (RAB1A) was a direct target of miR-655-3p in OC and was negatively regulated by miR-655-3p. Further, the effects of miR-655-3p/RAB1A axis on cell proliferation, metastasis ability, and EMT activation were emphasized. CONCLUSIONS: Our research emphasized the suppressor function of miR-655-3p in OC. By targeting RAB1A, miR-655-3p played a tumor suppressor role in OC. We affirmed the beneficial effects of miR-655-3p in OC cells for the first time, thus providing an experimental basis for the treatment of OC.

Inhibiting ERK/Mnk/eIF4E broadly sensitizes ovarian cancer response to chemotherapy

Purpose. To investigate whether ERK/MNK/eIF4E contributes chemoresistance in ovarian cancer. Methods. The phosphorylated levels of Erk, Mnk, and eIF4E were systematically analyzed in ovarian cancer patients before and after chemotherapy, and ovarian cancer cells exposed to short- and long-term chemo-agent treatment. The roles of Erk/Mnk/eIF4E were investigated using pharmacological and genetic approaches. Results. Increased phosphorylation levels of ERK, Mnk1, and eIF4E were observed in ovarian cancer cell exposed to chemotherapeutic agents, and paclitaxel-resistant SK-OV-3-r cells, and is a common response of ovarian cancer patients undergoing chemotherapy. MEK inhibitor U0126 inhibits basal and chemodrug-induced phosphorylation of ERK as well as Mnk1 and eIF4E, suggesting that Mnk1/eIF4E are the downstream signaling of ERK pathway and chemotherapy agents activate ERK/MNK/eIF4E in a MEK-dependent manner. eIF4E overexpression promotes ovarian cancer cell growth without affecting migration. In addition, ovarian cancer cells with eIF4E overexpression are more resistant to chemotherapeutic agents in aspect of growth inhibition and apoptosis induction compared to control cells. In contrast, eIF4E depletion augments chemotherapeutic agents’ effect in ovarian cancer cells. These demonstrate that eIF4E play roles in growth and chemoresistance in ovarian cancer. MEK inhibitor U0126 also significantly enhances chemotherapeutic agents’ inhibitory effects. Conclusions. Our work shows that ERK/Mnk/eIF4E activation is critically involved in ovarian cancer chemoresistance and inhibiting ERK/Mnk/eIF4E broadly sensitizes ovarian cancer response to chemotherapy (AU)

No disponible

Inhibiting ERK/Mnk/eIF4E broadly sensitizes ovarian cancer response to chemotherapy.

PURPOSE: To investigate whether ERK/MNK/eIF4E contributes chemoresistance in ovarian cancer. METHODS: The phosphorylated levels of Erk, Mnk, and eIF4E were systematically analyzed in ovarian cancer patients before and after chemotherapy, and ovarian cancer cells exposed to short- and long-term chemo-agent treatment. The roles of Erk/Mnk/eIF4E were investigated using pharmacological and genetic approaches. RESULTS: Increased phosphorylation levels of ERK, Mnk1, and eIF4E were observed in ovarian cancer cell exposed to chemotherapeutic agents, and paclitaxel-resistant SK-OV-3-r cells, and is a common response of ovarian cancer patients undergoing chemotherapy. MEK inhibitor U0126 inhibits basal and chemodrug-induced phosphorylation of ERK as well as Mnk1 and eIF4E, suggesting that Mnk1/eIF4E are the downstream signaling of ERK pathway and chemotherapy agents activate ERK/MNK/eIF4E in a MEK-dependent manner. eIF4E overexpression promotes ovarian cancer cell growth without affecting migration. In addition, ovarian cancer cells with eIF4E overexpression are more resistant to chemotherapeutic agents in aspect of growth inhibition and apoptosis induction compared to control cells. In contrast, eIF4E depletion augments chemotherapeutic agents' effect in ovarian cancer cells. These demonstrate that eIF4E play roles in growth and chemoresistance in ovarian cancer. MEK inhibitor U0126 also significantly enhances chemotherapeutic agents' inhibitory effects. CONCLUSIONS: Our work shows that ERK/Mnk/eIF4E activation is critically involved in ovarian cancer chemoresistance and inhibiting ERK/Mnk/eIF4E broadly sensitizes ovarian cancer response to chemotherapy.

Macrophage Transcriptional Profile Identifies Lipid Catabolic Pathways That Can Be Therapeutically Targeted after Spinal Cord Injury.

Although infiltrating macrophages influence many pathological processes after spinal cord injury (SCI), the intrinsic molecular mechanisms that regulate their function are poorly understood. A major hurdle has been dissecting macrophage-specific functions from those in other cell types as well as understanding how their functions change over time. Therefore, we used the RiboTag method to obtain macrophage-specific mRNA directly from the injured spinal cord in mice and performed RNA sequencing to investigate their transcriptional profile. Our data show that at 7 d after SCI, macrophages are best described as foam cells, with lipid catabolism representing the main biological process, and canonical nuclear receptor pathways as their potential mediators. Genetic deletion of a lipoprotein receptor, CD36, reduces macrophage lipid content and improves lesion size and locomotor recovery. Therefore, we report the first macrophage-specific transcriptional profile after SCI and highlight the lipid catabolic pathway as an important macrophage function that can be therapeutically targeted after SCI.SIGNIFICANCE STATEMENT The intrinsic molecular mechanisms that regulate macrophage function after spinal cord injury (SCI) are poorly understood. We obtained macrophage-specific mRNA directly from the injured spinal cord and performed RNA sequencing to investigate their transcriptional profile. Our data show that at 7 d after SCI, macrophages are best described as foam cells, with lipid catabolism representing the main biological process and canonical nuclear receptor pathways as their potential mediators. Genetic deletion of a lipoprotein receptor, CD36, reduces macrophage lipid content and improves lesion size and locomotor recovery. Therefore, we report the first macrophage-specific transcriptional profile after SCI and highlight the lipid catabolic pathway as an important macrophage function that can be therapeutically targeted after SCI.

[Prognostic significance of the changes of elastic fibers in acinar predominant or papillary-predominant pulmonary adenocarcinomas].

OBJECTIVE: To evaluate the role of elastic fiber changes in predicting survival outcomes in intermediate-grade lung adenocarcinoma. METHODS: All pulmonary adenocarcinoma resections conducted between January 2009 and December 2009 were reviewed. Pathologically confirmed adenocarcinomas smaller than 3 cm were included in the present study. All cases were categorized into three elastic fiber patterns (EFP): complete loss as pattern Ⅰ (EFP Ⅰ), partial loss as pattern Ⅱ (EFP Ⅱ), normal and diffusely increase as pattern Ⅲ (EFP Ⅲ). Patients with different EFP were compared. RESULTS: One hundred and ninety four patients were included in this study, with 67(34.5%), 70(36.1%)and 57(29.4%) cases presenting as EFP Ⅰ, EFP Ⅱ, and EFP Ⅲ, respectively. Lymph nodal metastases occurred in 35.8% (24/67), 40.0% (28/70), and 10.5% (6/57) of EFP Ⅰ, EFP Ⅱ and EFP Ⅲ patterns, respectively. The percentage of EFP Ⅰ and Ⅱ increased with increasing tumor size, these patterns occurring in 55.1% (38/69) of tumors ≤2.0 cm, and 79.2% (99/125) of tumors 2.1-3.0 cm in sizes, respectively. The overall 5-year overall survival rate was 75.8%, and 67.2% for EFP I, 68.6% for EFP II, and 94.7% for EFP Ⅲ. CONCLUSION: In patients with intermediate-grade lung adenocarcinoma, EFP should be formally recognized as a feature of tumor invasion, and its evaluation can help to recognize tumor invasive and access clinical prognosis.

Establishment of TBS reference plots and correlation between TBS and BMD in healthy mainland Chinese women.

UNLABELLED: The trabecular bone score (TBS) was obtained from the gray levels of a dual X-ray absorptiometry (DXA) image to evaluate bone microarchitecture. Here, we established the reference plots of TBS in healthy Chinese women of Nanjing area. The TBS references are similar with French and US Caucasian women but higher than Japanese women. PURPOSE: The aim of the study was to establish the reference plots of the TBS in healthy Chinese women of the Nanjing area. METHODS: A total of 537 healthy Chinese women of the Nanjing area were recruited, and the study was approved by the Ethics Committee of the First Affiliated Hospital with Nanjing Medical University. The TBS of the lumbar spine and the bone mineral density (BMD) of the lumbar spine and femur were measured using dual X-ray absorptiometry. The mean ± standard deviation (SD) of the TBS in women with different age groups was calculated. The correlation of TBS and age and BMD was calculated using regression analysis. RESULTS: The reference plots of the TBS were established in healthy Chinese women of the Nanjing area between the ages of 20 and 89 years. The average TBS for total subjects was 1.32 ± 0.11 and reached the peak at the age of 20-29 years and then decreased with age thereafter. The determinant coefficient between TBS and age was 0.5065 while between TBS and BMD was 0.5191. After adjusting for lumbar total BMD, the TBS significantly correlated with age in whole subjects and only in the subgroup of ages 50-59 years. CONCLUSIONS: This study suggested that TBS decreased with age and correlated positively with BMD. The TBS reference of Chinese women is similar with those of French and US Caucasian women but higher than that of Japanese women. Furthermore, the TBS may be more significantly applied in women in menopause for less than 10 years.

Establishment of reference range for thyroid hormones in normal pregnant women in China's coastal area.

PURPOSE: The current study aims to establish reference ranges for thyroid hormones in normal pregnant women during their pregnancy period. MATERIALS AND METHODS: A one-time cross-sectional survey was conducted on 490 normal pregnant women and 51 nonpregnant women (control). The serum thyroid stimulating hormone (TSH), free triiodothyronine (FT3), and free tetraiodothyronine (FT4) levels were measured. RESULTS: The serum FT3 and FT4 levels in pregnant women decreased gradually from the first to the last three months of pregnancy (p < 0.01). The serum TSH level increased gradually during the whole pregnancy (p < 0.01), and was significantly lower than the control (p < 0.01) in the first three months. However, in the middle and last three months of pregnancy, TSH was higher than the control (p < 0.01). CONCLUSIONS: The thyroid hormone levels in normal pregnant women are different from those in non-pregnant women; significant differences exist among the three stages of pregnancy.

Axl as a potential therapeutic target in cancer: role of Axl in tumor growth, metastasis and angiogenesis.

Dysregulation of Axl and its ligand growth arrest-specific 6 is implicated in the pathogenesis of several human cancers. In this study, we have used RNAi and monoclonal antibodies to assess further the oncogenic potential of Axl. Here we show that Axl knockdown reduces growth of lung and breast cancer xenograft tumors. Inhibition of Axl expression attenuates breast cancer cell migration and inhibits metastasis to the lung in an orthotopic model, providing the first in vivo evidence that links Axl directly to cancer metastasis. Axl knockdown in endothelial cells impaired tube formation and this effect was additive with anti-vascular endothelial growth factor (VEGF). Further analysis demonstrated that Axl regulates endothelial cell functions by modulation of signaling through angiopoietin/Tie2 and Dickkopf (DKK3) pathways. We have developed and characterized Axl monoclonal antibodies that attenuate non-small cell lung carcinoma xenograft growth by downregulation of receptor expression, reducing tumor cell proliferation and inducing apoptosis. Our data demonstrate that Axl plays multiple roles in tumorigenesis and that therapeutic antibodies against Axl may block Axl functions not only in malignant tumor cells but also in the tumor stroma. The additive effect of Axl inhibition with anti-VEGF suggests that blocking Axl function could be an effective approach for enhancing antiangiogenic therapy.

Functional characterization of the Bcl-2 gene family in the zebrafish.

Members of the Bcl-2 protein family control the intrinsic apoptosis pathway. To evaluate the importance of this family in vertebrate development, we investigated it in the zebrafish (Danio rerio). We found that the zebrafish genome encodes structural and functional homologs of most mammalian Bcl-2 family members, including multi-Bcl-2-homology (BH) domain proteins and BH3-only proteins. Apoptosis induction by gamma-irradiation required zBax1 and zPuma, and could be prevented by overexpression of homologs of prosurvival Bcl-2 family members. Surprisingly, zebrafish Bax2 (zBax2) was homologous to mammalian Bax by sequence and synteny, yet demonstrated functional conservation with human Bak. Morpholino knockdown of both zMcl-1a and zMcl-1b revealed their critical role in early embryonic zebrafish development, and in the modulation of apoptosis activation through the extrinsic pathway. These data indicate substantial functional similarity between zebrafish and mammalian Bcl-2 family members, and establish the zebrafish as a relevant model for studying the intrinsic apoptosis pathway.

Delineation of the cell-extrinsic apoptosis pathway in the zebrafish.

The mammalian extrinsic apoptosis pathway is triggered by Fas ligand (FasL) and Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL). Ligand binding to cognate receptors activates initiator caspases directly in a death-inducing signaling complex. In Drosophila, TNF ligand binding activates initiator caspases indirectly, through JNK. We characterized the extrinsic pathway in zebrafish to determine how it operates in a nonmammalian vertebrate. We identified homologs of FasL and Apo2L/TRAIL, their receptors, and other components of the cell death machinery. Studies with three Apo2L/TRAIL homologs demonstrated that they bind the receptors zHDR (previously linked to hematopoiesis) and ovarian TNFR (zOTR). Ectopic expression of these ligands during embryogenesis induced apoptosis in erythroblasts and notochord cells. Inhibition of zHDR, zOTR, the adaptor zFADD, or caspase-8-like proteases blocked ligand-induced apoptosis, as did antiapoptotic Bcl-2 family members. Thus, the extrinsic apoptosis pathway in zebrafish closely resembles its mammalian counterpart and cooperates with the intrinsic pathway to trigger tissue-specific apoptosis during embryogenesis in response to ectopic Apo2L/TRAIL expression.

Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis.

Many apoptotic molecules relocate subcellularly in cells undergoing apoptosis. The pro-apoptotic protein BID underwent posttranslational (rather than classic cotranslational) N-myristoylation when cleavage by caspase 8 caused exposure of a glycine residue. N-myristoylation enabled the targeting of a complex of p7 and myristoylated p15 fragments of BID to artificial membranes bearing the lipid composition of mitochondria, as well as to intact mitochondria. This post-proteolytic N-myristoylation serves as an activating switch, enhancing BID-induced release of cytochrome c and cell death.

Differential modulation of apoptosis sensitivity in CD95 type I and type II cells.

We have recently identified two different pathways of CD95-mediated apoptosis (Scaffidi, C., Fulda, S., Srinivasan, A., Feng, L., Friesen, C., Tomaselli, K. J., Debatin, K.-M., Krammer, P. H., and Peter, M. E. (1998) EMBO J. 17, 1675-1687). CD95-mediated apoptosis in type I cells is initiated by large amounts of active caspase-8 formed at the death-inducing signaling complex (DISC) followed by direct cleavage of caspase-3. In contrast, in type II cells very little DISC and small amounts of active caspase-8 sufficient to induce the apoptogenic activity of mitochondria are formed causing a profound activation of both caspase-8 and caspase-3. Only in type II cells can apoptosis be blocked by overexpressed Bcl-2 or Bcl-x(L). We now show that a number of apoptosis-inhibiting or -inducing stimuli only affect apoptosis in type II cells, indicating that they act on the mitochondrial branch of the CD95 pathway. These stimuli include the activation of protein kinase C, which inhibits CD95-mediated apoptosis resulting in a delayed cleavage of BID, and the induction of apoptosis by the ceramide analog C(2)-ceramide. In addition, we have identified the CD95 high expressing cell line Boe(R) as a CD95 apoptosis-resistant type II cell that can be sensitized by treatment with cycloheximide without affecting formation of the DISC. This also places the effects of cycloheximide in the mitochondrial branch of the type II CD95 pathway. In contrast, c-FLIP was found to block CD95-mediated apoptosis in both type I and type II cells, because it acts directly at the DISC of both types of cells.

Trophic support promotes survival of bcl-x-deficient telencephalic cells in vitro.

Survival of immature neurons is regulated by Bcl-xL, as targeted disruption of bcl-x significantly increases cell death in vivo and in vitro. Death of cultured bcl-x-deficient and wild-type telencephalic cells can be prevented by fetal calf serum or chemically-defined medium (ITS), suggesting trophic factors in these media potentiate survival through a pathway independent of Bcl-xL. Addition of trophic factors to basal medium revealed that insulin and insulin-like growth factors (IGFs), but not other trophic factors, reduced apoptosis of wild-type and bcl-x-deficient telencephalic cells. Antibodies raised against IGF-I receptors and wortmannin both attenuated the effects of IGF-I, indicating survival was mediated by IGF-I receptors and phosphatidylinositol 3'-kinase signaling, whereas effects of ITS were only partially reduced by these agents. The survival promoting effects of ITS were reduced in cells lacking both bcl-x and bcl-2, indicating Bcl-2 plays a supportive role to Bcl-xL in maintaining telencephalic cell survival. Furthermore, the ratio of expression of the pro-apoptotic bax gene to the anti-apoptotic bcl-2 gene was reduced in bcl-x-deficient cultures grown in ITS, suggesting that the interaction between these bcl-2 family members may, in part, regulate a Bcl-xL independent survival pathway. Finally, the pro-apoptotic bad gene does not appear to play a role in these interactions as targeted disruption of bad did not alter apoptosis in telencephalic cultures.

[Cetyltrimethylammonium bromide fluorescence enhancement of aspirin].

The fluorescence intensities of aspirin with cetyltrimethylammonium bromide enhanced by micellar solutions have been examined. It is found that fluorescence enhancement depend on the concentration of CTAB and pH of solution.