A multicentre, open-label, phase-I/randomised phase-II study to evaluate safety, pharmacokinetics, and efficacy of nintedanib vs. sorafenib in European patients with advanced hepatocellular carcinoma.

BACKGROUND: This multicentre, open-label, phase-I/randomised phase-II trial evaluated safety, pharmacokinetics, maximum-tolerated-dose (MTD) per dose-limiting toxicities (DLTs), and efficacy of nintedanib vs. sorafenib in European patients with unresectable advanced hepatocellular carcinoma (aHCC). METHODS: Phase I: Patients were stratified into two groups per baseline aminotransferase/alanine aminotransferase and Child-Pugh score; MTD was determined. Phase II: Patients were randomised 2:1 to nintedanib (MTD) or sorafenib (400-mg bid) in 28-day cycles until intolerance or disease progression. Time-to-progression (TTP, primary endpoint), overall survival (OS) and progression-free survival (PFS) were determined. RESULTS: Phase-I: no DLTs observed; nintedanib MTD in both groups was 200 mg bid. Phase-II: patients (N = 93) were randomised to nintedanib (n = 62) or sorafenib (n = 31); TTP was 5.5 vs. 4.6 months (HR = 1.44 [95% CI, 0.81-2.57]), OS was 11.9 vs. 11.4 months (HR = 0.88 [95% CI, 0.52-1.47]), PFS was 5.3 vs. 3.9 months (HR = 1.35 [95% CI, 0.78-2.34]), respectively (all medians). Dose intensity and tolerability favoured nintedanib. Fewer patients on nintedanib (87.1%) vs. sorafenib (96.8%) had drug-related adverse events (AEs) or grade ≥ 3 AEs (67.7% vs. 90.3%), but more patients on nintedanib (28 [45.2%]) had AEs leading to drug discontinuation than did those on sorafenib (7 [22.6%]). CONCLUSIONS: Nintedanib may have similar efficacy to sorafenib in aHCC.

Significance of herpesvirus 6 in BAL fluid of hematology patients with acute respiratory failure.

PURPOSE: Human herpesvirus 6 (HHV6) is an emerging cause of interstitial pneumonia in immunocompromised hosts. However, the clinical significance of a positive PCR test for HHV6 in respiratory samples from patients with hematological malignancies remains unclear. METHODS: We retrospectively studied the features and outcomes of 29 critically ill hematology patients with acute respiratory failure and lung pulmonary infiltrates visible on a chest radiograph, who tested positive for a qualitative PCR for HHV6 in bronchoalveolar lavage fluid. RESULTS: Of the 29 patients, 18 (62%) were stem cell transplant recipients and 11 (38%) had received chemotherapy. All patients had a fever. Clinical manifestations consistent with extra-pulmonary HHV6 disease were noted in 17 (59%) patients. One or more co-pathogens were found in 25 (86%) patients. The four remaining patients diagnosed with HHV6 pneumonia and subsequently recovered with foscarnet therapy. Antiviral therapy was also given to seven patients with co-infections, of whom two ultimately died. CONCLUSIONS: In most cases, HHV6 recovered from BAL fluid is a co-pathogen whose clinical relevance remains undetermined. However, in some cases, HHV6 is the only pathogen, along with disseminated systemic viral disease, and the patient is likely to benefit from foscarnet therapy.

Protein import into chloroplasts.

Chloroplasts have evolved an elaborate system of membrane and soluble subcompartments to organize and regulate photosynthesis and essential aspects of amino acid and lipid metabolism. The biogenesis and maintenance of organellar architecture rely on protein subunits encoded by both nuclear and plastid genomes. Import of nuclear-encoded proteins is mediated by interactions between the intrinsic N-terminal transit sequence of the nuclear-encoded preprotein and a common import machinery at the chloroplast envelope. Recent investigations have shown that there are two unique membrane-bound translocation systems, in the outer and inner envelope membranes, which physically associate during import to transport preproteins from the cytoplasm to the internal stromal compartment. This review discusses current understanding of these translocation systems and models for the way in which they might function.

Identification of intermediates in the pathway of protein import into chloroplasts and their localization to envelope contact sites.

We have used a hybrid precursor protein to study the pathway of protein import into chloroplasts. This hybrid (pS/protA) consists of the precursor to the small subunit of Rubisco (pS) fused to the IgG binding domains of staphylococcal protein A. The pS/protA is efficiently imported into isolated chloroplasts and is processed to its mature form (S/protA). In addition to the mature stromal form, two intermediates in the pathway of pS/protA import were identified at early time points in the import reaction. The first intermediate represents unprocessed pS/protA bound to the outer surface of the chloroplast envelope and is analogous to a previously characterized form of pS that is specifically bound to the chloroplast surface and can be subsequently translocated in the stroma (Cline, K., M. Werner-Washburne, T. H. Lubben, and K. Keegstra. 1985. J. Biol. Chem. 260:3691-3696.) The second intermediate represents a partially translocated form of the precursor that remains associated with the envelope membrane. This form is processed to mature S/protA, but remains susceptible to exogenously added protease in intact chloroplasts. We conclude that the envelope associated S/protA is spanning both the outer and inner chloroplast membranes en route to the stroma. Biochemical and immunochemical localization of the two translocation intermediates indicates that both forms are exposed at the surface of the outer membrane at sites where the outer and inner membrane are closely apposed. These contact zones appear to be organized in a reticular network on the outer envelope. We propose a model for protein import into chloroplasts that has as its central features two distinct protein conducting channels in the outer and inner envelope membranes, each gated open by a distinct subdomain of the pS signal sequence.

Analysis of the interactions of preproteins with the import machinery over the course of protein import into chloroplasts.

We have investigated the interactions of two nuclear-encoded preproteins with the chloroplast protein import machinery at three stages in import using a label-transfer crosslinking approach. During energy-independent binding at the outer envelope membrane, preproteins interact with three known components of the outer membrane translocon complex, Toc34, Toc75, and Toc86. Although Toc75 and Toc86 are known to associate with preproteins during import, a role for Toc34 in preprotein binding previously had not been observed. The interaction of Toc34 with preproteins is regulated by the binding, but not hydrolysis of GTP. These data provide the first evidence for a direct role for Toc34 in import, and provide insights into the function of GTP as a regulator of preprotein recognition. Toc75 and Toc86 are the major targets of cross-linking upon insertion of preproteins across the outer envelope membrane, supporting the proposal that both proteins function in translocation at the outer membrane as well as preprotein recognition. The inner membrane proteins, Tic(21) and Tic22, and a previously unidentified protein of 14 kD are the major targets of crosslinking during the late stages in import. These data provide additional support for the roles of these components during protein translocation across the inner membrane. Our results suggest a defined sequence of molecular interactions that result in the transport of nuclear-encoded preproteins from the cytoplasm into the stroma of chloroplasts.

The chloroplast import receptor is an integral membrane protein of chloroplast envelope contact sites.

A chloroplast import receptor from pea, previously identified by antiidiotypic antibodies was purified and its primary structure deduced from its cDNA sequence. The protein is a 36-kD integral membrane protein (p36) with eight potential transmembrane segments. Fab prepared from monospecific anti-p36 IgG inhibits the import of the ribulose-1,5-bisphosphate carboxylase small subunit precursor (pS) by interfering with pS binding at the chloroplast surface. Anti-p36 IgGs are able to immunoprecipitate a Triton X-100 soluble p36-pS complex, suggesting a direct interaction between p36 and pS. This immunoprecipitation was specific as it was abolished by a pS synthetic transit peptide, consistent with the transit sequence receptor function of p36. Immunoelectron microscopy localized p36 to regions of the outer chloroplast membrane that are in close contact with the inner chloroplast membrane. Comparison of the deduced sequence of pea p36 to that of other known proteins indicates a striking homology to a protein from spinach chloroplasts that was previously suggested to be the triose phosphate-3-phosphoglycerate-phosphate translocator (phosphate translocator) (Flügge, U. I., K. Fischer, A. Gross, W. Sebald, F. Lottspeich, and C. Eckerskorn. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:39-46). However, incubation of Triton X-100 solubilized chloroplast envelope material with hydroxylapatite indicated that p36 was quantitatively absorbed, whereas previous reports have shown that phosphate translocator activity does not bind to hydroxylapatite (Flügge, U. I., and H. W. Heldt. 1981. Biochim. Biophys. Acta. 638:296-304. These data, in addition to the topology and import inhibition data presented in this report support the assignment of p36 as a receptor for chloroplast protein import, and argue against the assignment of the spinach homologue of this protein as the chloroplast phosphate translocator.

Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane.

Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677-1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.

Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope.

The interactions of precursor proteins with components of the chloroplast envelope were investigated during the early stages of protein import using a chemical cross-linking strategy. In the absence of energy, two components of the outer envelope import machinery, IAP86 and IAP75, cross-linked to the transit sequence of the precursor to the small subunit of ribulose-1, 5-bisphosphate carboxylase (pS) in a precursor binding assay. In the presence of concentrations of ATP or GTP that support maximal precursor binding to the envelope, cross-linking to the transit sequence occurred predominantly with IAP75 and a previously unidentified 21-kD polypeptide of the inner membrane, indicating that the transit sequence had inserted across the outer membrane. Cross-linking of envelope components to sequences in the mature portion of a second precursor, preferredoxin, was detected in the presence of ATP or GTP, suggesting that sequences distant from the transit sequence were brought into the vicinity of the outer membrane under these conditions. IAP75 and a third import component, IAP34, were coimmunoprecipitated with IAP86 antibodies from solubilized envelope membranes, indicating that these three proteins form a stable complex in the outer membrane. On the basis of these observations, we propose that IAP86 and IAP75 act as components of a multisubunit complex to mediate energy-independent recognition of the transit sequence and subsequent nucleoside triphosphate-induced insertion of the transit sequence across the outer membrane.

Isolation of components of the chloroplast protein import machinery.

Components of the protein import machinery of the chloroplast were isolated by a procedure in which the import machinery was engaged in vitro with a tagged import substrate under conditions that yielded largely chloroplast envelope-bound import intermediates. Subsequent detergent solubilization of envelope membranes showed that six envelope polypeptides copurified specifically and, apparently, stoichiometrically with the import intermediates. Four of these polypeptides are components of the outer membrane import machinery and are associated with early import intermediates. Two of these polypeptides have been characterized. One is a homolog of the heat shock protein hsp70; the other one is a channel-protein candidate.

Identification of two GTP-binding proteins in the chloroplast protein import machinery.

Two of four proteins that associated with translocation intermediates during protein import across the outer chloroplast envelope membrane were identified as guanosine triphosphate (GTP)-binding proteins. Both proteins are integral membrane proteins of the outer chloroplast membrane, and both are partially exposed on the chloroplast surface where they were accessible to thermolysin digestion. Engagement of the outer membrane's import machinery by an import substrate was inhibited by slowly hydrolyzable or non-hydrolyzable GTP analogs. Thus, these GTP-binding proteins may function in protein import into chloroplasts.

Bioassay of solubilized Bacillus thuringiensis var. israelensis crystals by attachment to latex beads.

Solubilized crystals of Bacillus thuringiensis var. israelensis were 7000 times less toxic to Aedes aegypti larvae than intact crystals, presumably because mosquito larvae are filter feeders and selectively concentrate particles while excluding water and soluble molecules. A procedure is described whereby soluble toxins are adsorbed to 0.8-micrometer latex beads, with retention of toxicity. The latex bead assay should make it possible to analyze the structure and mode of action of the mosquito toxin.

Manganese superoxide dismutase deficiency triggers mitochondrial uncoupling and the Warburg effect.

This corrects the article DOI: 10.1038/onc.2014.355.

Antiarrhythmic effects of azimilide in atrial fibrillation: efficacy and dose-response. Azimilide Supraventricular Arrhythmia Program 3 (SVA-3) Investigators.

OBJECTIVES: The purpose of this study was to assess the effectiveness of azimilide, a class III antiarrhythmic drug, in reducing the frequency of symptomatic arrhythmia recurrences in patients with atrial fibrillation, atrial flutter or both. BACKGROUND: Atrial fibrillation is an increasingly common disorder of the heart rhythm, and most patients with this problem are identified because they have symptoms associated with their arrhythmia. New antiarrhythmic therapies are needed to treat patients with this problem. METHODS: A total of 384 patients with a history of atrial fibrillation, atrial flutter or both were randomly assigned to receive once daily doses of placebo or azimilide; recurrent symptomatic arrhythmias were documented using transtelephonic electrocardiogram (ECG) recording. Azimilide 50 mg, 100 mg or 125 mg was tested; the primary efficacy analysis compared the time to first symptomatic recurrence in the combined azimilide 100 mg and 125 mg dose groups with that in the placebo group using the log-rank test. RESULTS: In the primary efficacy analysis, the time to first symptomatic arrhythmia recurrence was significantly prolonged in the combined azimilide 100 mg and 125 mg daily dose group compared with the placebo group (chi-square 7.96, p = 0.005); the hazard ratio (placebo: azimilide) for this comparison was 1.58 (95% confidence interval [CI] = 1.15, 2.16). In comparisons between individual doses and placebo, the hazard ratio for the 50 mg daily dose was 1.17 (95% CI = 0.83, 1.66; p = 0.37); for the 100 mg group, dose was 1.38 (95% CI = 0.96, 1.98; p = 0.08), and for the 125 mg group, dose was 1.83 (95% CI = 1.24, 2.70; p = 0.002). CONCLUSIONS: Azimilide significantly lengthened the symptomatic arrhythmia-free interval in patients with a history of atrial fibrillation, atrial flutter or both.

Functions and origins of the chloroplast protein-import machinery.

The vast majority of chloroplast proteins are nuclear-encoded and are imported into the organelle after synthesis in the cytoplasm. Targeting to chloroplasts is mediated by a variety of intrinsic targeting signals that direct the preprotein to its proper organelle subcompartment. Translocation at the envelope membrane is directed by the interactions of an N-terminal transit sequences on the preprotein and a general import machinery composed of the outer-membrane Toc machinery and the inner-membrane Tic machinery. The Toc and Tic components interact to bypass the intermembrane space and provide direct transport of preproteins from the cytoplasm to the stroma. There are at least four targeting pathways to the thylakoid membrane, the cpSec pathway, the delta pH pathway, the cpSRP pathway and the spontaneous pathway. These pathways require distinct intrinsic targeting signals, and apparently evolved to accommodate the translocation of classes of proteins with particular characteristics. Proteins similar to some components of the envelope and thylakoid translocation pathways are found in bacterial systems. However, a number of components do not have bacterial counterparts and are unique to the chloroplast pathways. It therefore appears that the chloroplast translocation systems have evolved from membrane-transport systems that were present in the original endosymbiont by incorporating proteins necessary to adapt to the constraints of endosymbiosis.

[Lymph node dissection in patients with renal cell carcinoma]. / Lymphknotendissektion beim Nierenzellkarzinom.

The role of lymph node dissection in renal cell carcinoma has been controversial for decades. However, the results of the only prospective randomised study concerning this issue (EORTC 30881) are preliminary. Retrospective analyses were not able to demonstrate a diagnostic or therapeutic benefit of an extended lymph node dissection. Suspicious lymph nodes (imaging, palpation) should be excised during nephrectomy.

[Patterns of care of patients with localized prostate cancer in Germany: a health care study with focus on active surveillance]. / Die Versorgung von Patienten mit lokal begrenztem Prostatakarzinom in Deutschland : Active-Surveillance-Ergebnisse einer nicht-interventionellen Versorgungsstudie.

BACKGROUND: To date, evidence on active surveillance (AS) is restricted to protocol-based studies and the current practice pattern outside medical centers is unknown. OBJECTIVES: The goal of this work was to capture the current treatment pattern of AS for localized prostate cancer (PCa) in patients managed by office-based urologists in Germany. MATERIALS AND METHODS: Our cohort consisted of 361 patients included in the AS arm of the HAROW (Hormonal Treatment, Active Surveillance, Radiation Therapy, OP, Watchful Waiting) study, an observational health service study in Germany. Descriptive characteristics and active-treatment-free survival (ATFS), surgical outcomes, and triggers for active treatment were assessed. RESULTS: Currently, only 15% of all patients with localized PCa were treated with AS. At baseline, 83% and 58% of all AS patients met the Chism and PRIAS low-risk criteria, respectively. After a median follow-up of 24 months, no systemic progression was observed, 5 patients died of non-disease-specific causes and active treatment was delivered in 20.5% of all patients. Triggers for active therapy were progression at biopsy (42%), rise in prostate-specific antigen level (27%), medical advice (16%) and patient's preference (10%), respectively. CONCLUSION: Our short-term results indicate that - in the hands of office-based urologists - active surveillance might represent a feasible treatment option for patients with localized PCa. The majority of patients were free of active treatment 2 years after AS initiation. Generally accepted inclusion and progression criteria are lacking and should be developed in order to facilitate and standardize AS in patients with low-risk PCa.

Life-threatening complications and outcomes in patients with malignancies and severe pulmonary embolism.

BACKGROUND: Data are scarce about ICU patients with malignancy and severe pulmonary embolism. Here, our main objective was to identify risk factors for life-threatening complications, organ failures, and death in ICU patients with severe pulmonary embolism, with special attention to the impact of malignancy. We also described the clinical features of PE in patients with and without malignancies. METHODS: Data from consecutive adults admitted to our ICU in 2002-2011 with severe pulmonary embolism were collected retrospectively. Multivariate analysis was performed to look for factors associated with death, organ failures, or life-threatening complications (major bleeding, recurrent PE, and cardiac arrest). RESULTS: Of 119 included patients (42 [35%] with bilateral pulmonary embolism), 41 had solid malignancies, 27 hematological malignancies, and 51 no malignancies. The most common symptoms were syncope (40%) and hemoptysis (18%) in patients with solid and hematological malignancies, respectively. Life-threatening complications occurred in 23 (19%) patients; risk factors were obesity (OR, 13.22; 1.93-90.70), disseminated intravascular coagulation/ischemic hepatitis (OR, 27.06; 5.14-142.46), fluid load ≥1000 mL/24 h (OR, 6.42; 1.60-25.76), and solid malignancy (OR, 5.45; 1.15-25.89). Inhospital mortality was 27/119 (23%) and respiratory or circulatory failure developed in 36 (30%) patients. Risk factors for these adverse outcomes were older age (OR, 1.04/year; 1.01-1.07), higher oxygen flow rate (OR, 1.28/L; 1.13-1.45); and renal failure (OR, 8.08; 2.50-26.11); whereas chest pain was protective (OR, 0.13; 0.04-0.48). CONCLUSION: In this study, solid malignancy was a risk factor for life-threatening complications but not for death.

Manganese superoxide dismutase deficiency triggers mitochondrial uncoupling and the Warburg effect.

Manganese superoxide dismutase (MnSOD) is a mitochondrially localized primary antioxidant enzyme, known to be essential for the survival of aerobic life and to have important roles in tumorigenesis. Here, we show that MnSOD deficiency in skin tissues of MnSOD-heterozygous knockout (Sod2(+/-)) mice leads to increased expresson of uncoupling proteins (UCPs). When MnSOD is deficient, superoxide radical and its resulting reactive oxygen species (ROS) activate ligand binding to peroxisome proliferator-activated receptor alpha (PPARα), suggesting that the activation of PPARα signaling is a major mechanism underlying MnSOD-dependent UCPs expression that consequently triggers the PI3K/Akt/mTOR pathway, leading to increased aerobic glycolysis. Knockdown of UCPs and mTOR suppresses lactate production and increases ATP levels, suggesting that UCPs contribute to increased glycolysis. These results highlight the existence of a free radical-mediated mechanism that activates mitochondria uncoupling to reduce ROS production, which precedes the glycolytic adaptation described as the Warburg Effect.

A phase I study of daily afatinib, an irreversible ErbB family blocker, in combination with weekly paclitaxel in patients with advanced solid tumours.

BACKGROUND: This phase I study evaluated afatinib, an irreversible ErbB family blocker, plus paclitaxel in patients with advanced solid tumours likely to express human epidermal growth factor receptor (HER1/EGFR) or HER2. METHODS: Oral afatinib was combined with intravenous paclitaxel (80mg/m(2); days 1, 8 and 15 every four weeks) starting at 20mg once daily and escalated to 40 and 50mg in successive cohorts of ⩾3 patients. The primary objective was to determine the maximum tolerated dose (MTD) of afatinib combined with paclitaxel. Secondary objectives included safety, pharmacokinetics and antitumour activity. RESULTS: Sixteen patients were treated. Dose-limiting toxicities with afatinib 50mg were fatigue and mucositis. The MTD was determined as afatinib 40mg with paclitaxel 80mg/m(2), which proved tolerable with repeated dosing. Frequent adverse events (AEs) included diarrhoea (94%), fatigue (81%), rash/acne (81%), decreased appetite (69%) and inflammation of mucosal membranes (69%); no grade 4 treatment-related AEs were observed. Five (31%) confirmed partial responses were observed in patients with non-small cell lung cancer (n=3), oesophageal cancer and cholangiocarcinoma; eight (50%) patients remained on study for ⩾6months. Pharmacokinetic parameters of afatinib and paclitaxel were similar for single administration or in combination. CONCLUSIONS: The MTD and recommended phase II dose of once-daily afatinib combined with paclitaxel 80mg/m(2) (days 1, 8 and 15 every four weeks) was 40mg. AEs at or below this dose were generally manageable with repeated dosing. No pharmacokinetic interactions were observed. This combination demonstrated promising antitumour activity. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00809133.

Dose-response relations of azimilide in the management of symptomatic, recurrent, atrial fibrillation.

We evaluated the efficacy and safety of azimilide, a new class III antiarrhythmic agent that blocks both the slow and fast components of the cardiac-delayed rectifier potassium currents in 4 randomized, double-blind, placebo-controlled trials with similar protocols. The purpose of this study was to assess the relation between dose and effect. A total of 1,380 patients with a documented history of symptomatic atrial fibrillation (AF), atrial flutter, or both, were enrolled. After a 3-day loading period during which the assigned dose was given twice a day, subjects received placebo or azimilide (35, 50, 75, 100, or 125 mg once a day) for the duration of the study period. The primary end point of the studies was the time to symptomatic arrhythmia recurrence with a transtelephonic electrocardiogram typical of AF, atrial flutter, or paroxysmal supraventricular tachycardia. For each study, Kaplan-Meier estimates of the median time to recurrence were computed for placebo and for each azimilide dose. Cox proportional-hazards modeling was used to estimate hazard ratios for each active dose. Each of the 2 highest azimilide doses (100 and 125 mg/day) significantly prolonged the time to recurrence of arrhythmia. For the 100 mg/day dose, the hazard ratio was 1.34, 95% confidence interval 1.05 to 1.72; p = 0.02. For the 125 mg/day dose, the hazard ratio was 1.32, 95% confidence interval 1.07 to 1.62; p = 0.01. Patients with a history of either ischemic heart disease or congestive heart failure had a significantly greater treatment effect from azimilide than those without it. Torsades de Pointes occurred in 0.9% of patients receiving either of the 2 effective doses. Thus, doses of azimilide <100 mg/day are not effective for control of AF, whereas doses of 100 and 125 mg/day are effective with an acceptable risk of serious toxicity.