Determination of four sequential stages during microautophagy in vitro.

Microautophagy is the transfer of cytosolic components into the lysosome by direct invagination of the lysosomal membrane and subsequent budding of vesicles into the lysosomal lumen. This process is topologically equivalent to membrane invagination during multivesicular body formation and to the budding of enveloped viruses. Vacuoles are lysosomal compartments of yeasts. Vacuolar membrane invagination can be reconstituted in vitro with purified yeast vacuoles, serving as a model system for budding of vesicles into the lumen of an organelle. Using this in vitro system, we defined different reaction states. We identified inhibitors of microautophagy in vitro and used them as tools for kinetic analysis. This allowed us to characterize four biochemically distinguishable steps of the reaction. We propose that these correspond to sequential stages of vacuole invagination and vesicle scission. Formation of vacuolar invaginations was slow and temperature-dependent, whereas the final scission of the vesicle from a preformed invagination was fast and proceeded even on ice. Our observations suggest that the formation of invaginations rather than the scission of vesicles is the rate-limiting step of the overall reaction.

A Reply to comments by D. Bar-Yosef M. Stiner on "Prehistoric exploitation of marine resources in Southern Africa with particular reference to shellfish gathering: opportunities and continuities"

I am very grateful to Daniella E. Bar-Yosef Mayer and Mary Stiner for their comments on an article I published in the previous issue of Pyrenae. Having spent many years working with coastal sites in South Africa and now settling in the Mediterranean academic landscape, I value the feedback from these two well-known archaeologists who have dedicated years of hard work in this later part of the world. Their opinions are very much appreciated for they allow me to bring new contexts to some of the (old) questions I have pursued in South Africa, an exercise that would help me with the process of broadening my research interests to the Mediterranean region.

Enurésie nocturne : quel traitement efficace non pharmacologique recommander ?

L'énurésie nocturne est une affection commune qui touche jusqu'à 20% des enfants de 5 ans. Cette prévalence diminue ensuite rapidement pour atteindre 7% à l'âge de 7 ans et pas plus de 2% à l'âge adulte. Quoique bénigne, elle est souvent source de stigmatisation sociale et peut affecter à la fois l'estime de soi et les relations avec les pairs. Les interventions de type comportemental (ICS) sont fréquentes et constituent souvent le premier choix des parents et de l'enfant. Une controverse persiste toutefois quant à leur efficacité. Le but de la mise à jour de cette revue est d'évaluer l'efficacité d'interventions comportementales simples (ICS : restriction hydrique, lever, réveil, récompenses, exercices de rétention), entre elles et par rapport à d'autres traitements.

Impact of tight glycemic control on cerebral glucose metabolism after severe brain injury: a microdialysis study.

OBJECTIVES: To analyze the effect of tight glycemic control with the use of intensive insulin therapy on cerebral glucose metabolism in patients with severe brain injury. DESIGN: Retrospective analysis of a prospective observational cohort. SETTING: University hospital neurologic intensive care unit. PATIENTS: Twenty patients (median age 59 yrs) monitored with cerebral microdialysis as part of their clinical care. INTERVENTIONS: Intensive insulin therapy (systemic glucose target: 4.4-6.7 mmol/L [80-120 mg/dL]). MEASUREMENTS AND MAIN RESULTS: Brain tissue markers of glucose metabolism (cerebral microdialysis glucose and lactate/pyruvate ratio) and systemic glucose were collected hourly. Systemic glucose levels were categorized as within the target "tight" (4.4-6.7 mmol/L [80-120 mg/dL]) vs. "intermediate" (6.8-10.0 mmol/L [121-180 mg/dL]) range. Brain energy crisis was defined as a cerebral microdialysis glucose <0.7 mmol/L with a lactate/pyruvate ratio >40. We analyzed 2131 cerebral microdialysis samples: tight systemic glucose levels were associated with a greater prevalence of low cerebral microdialysis glucose (65% vs. 36%, p < 0.01) and brain energy crisis (25% vs.17%, p < 0.01) than intermediate levels. Using multivariable analysis, and adjusting for intracranial pressure and cerebral perfusion pressure, systemic glucose concentration (adjusted odds ratio 1.23, 95% confidence interval [CI] 1.10-1.37, for each 1 mmol/L decrease, p < 0.001) and insulin dose (adjusted odds ratio 1.10, 95% CI 1.04-1.17, for each 1 U/hr increase, p = 0.02) independently predicted brain energy crisis. Cerebral microdialysis glucose was lower in nonsurvivors than in survivors (0.46 +/- 0.23 vs. 1.04 +/- 0.56 mmol/L, p < 0.05). Brain energy crisis was associated with increased mortality at hospital discharge (adjusted odds ratio 7.36, 95% CI 1.37-39.51, p = 0.02). CONCLUSIONS: In patients with severe brain injury, tight systemic glucose control is associated with reduced cerebral extracellular glucose availability and increased prevalence of brain energy crisis, which in turn correlates with increased mortality. Intensive insulin therapy may impair cerebral glucose metabolism after severe brain injury.

Tadpole pupil.

Editors' Note: Dr. Egan inquires about the possibility of Tournay phenomenon in Kawasaki and Mayer's "Tadpole pupil." Dr. Charles, in reference to "Disease-modifying drugs for multiple sclerosis in pregnancy: A systematic review," suggests that it might be appropriate to continue glatiramer acetate in women trying to conceive given the lack of evidence of fetal risk. Authors Tremlett et al. advise that, at this point, each case should continue to be assessed individually.

Role of the V-ATPase in regulation of the vacuolar fission-fusion equilibrium.

Like numerous other eukaryotic organelles, the vacuole of the yeast Saccharomyces cerevisiae undergoes coordinated cycles of membrane fission and fusion in the course of the cell cycle and in adaptation to environmental conditions. Organelle fission and fusion processes must be balanced to ensure organelle integrity. Coordination of vacuole fission and fusion depends on the interactions of vacuolar SNARE proteins and the dynamin-like GTPase Vps1p. Here, we identify a novel factor that impinges on the fusion-fission equilibrium: the vacuolar H(+)-ATPase (V-ATPase) performs two distinct roles in vacuole fission and fusion. Fusion requires the physical presence of the membrane sector of the vacuolar H(+)-ATPase sector, but not its pump activity. Vacuole fission, in contrast, depends on proton translocation by the V-ATPase. Eliminating proton pumping by the V-ATPase either pharmacologically or by conditional or constitutive V-ATPase mutations blocked salt-induced vacuole fragmentation in vivo. In living cells, fission defects are epistatic to fusion defects. Therefore, mutants lacking the V-ATPase display large single vacuoles instead of multiple smaller vacuoles, the phenotype that is generally seen in mutants having defects only in vacuolar fusion. Its dual involvement in vacuole fission and fusion suggests the V-ATPase as a potential regulator of vacuolar morphology and membrane dynamics.

Characterisation of the PSI whole body counter by radiographic imaging.

A joint project between the Paul Scherrer Institut (PSI) and the Institute of Radiation Physics was initiated to characterise the PSI whole body counter in detail through measurements and Monte Carlo simulation. Accurate knowledge of the detector geometry is essential for reliable simulations of human body phantoms filled with known activity concentrations. Unfortunately, the technical drawings provided by the manufacturer are often not detailed enough and sometimes the specifications do not agree with the actual set-up. Therefore, the exact detector geometry and the position of the detector crystal inside the housing were determined through radiographic images. X-rays were used to analyse the structure of the detector, and (60)Co radiography was employed to measure the core of the germanium crystal. Moreover, the precise axial alignment of the detector within its housing was determined through a series of radiographic images with different incident angles. The hence obtained information enables us to optimise the Monte Carlo geometry model and to perform much more accurate and reliable simulations.

Combination of bevacizumab and 2-weekly pegylated liposomal doxorubicin as first-line therapy for locally recurrent or metastatic breast cancer. A multicenter, single-arm phase II trial (SAKK 24/06).

BACKGROUND: Pegylated liposomal doxorubicin (PLD) and bevacizumab are active agents in the treatment of metastatic breast cancer (MBC). We carried out a multicenter, single-arm phase II trial to evaluate the toxicity and efficacy of PLD and bevacizumab as first-line treatment in MBC patients. METHODS: Bevacizumab (10 mg/kg) and PLD (20 mg/m(2)) were infused on days 1 and 15 of a 4-week cycle for a maximum of six cycles. Thereafter, bevacizumab monotherapy was continued at the same dose until progression or toxicity. The primary objective was safety and tolerability, and the secondary objective was to evaluate efficacy of the combination. RESULTS: Thirty-nine of 43 patients were assessable for the primary end point. Eighteen of 39 patients (46%, 95% confidence interval 30% to 63%) had a grade 3 toxicity. Sixteen (41%) had grade 3 palmar-plantar erythrodysesthesia, one had grade 3 mucositis, and one severe cardiotoxicity. Secondary end point of overall response rate among 43 assessable patients was 21%. CONCLUSIONS: In this nonrandomized single-arm trial, the combination of bimonthly PLD and bevacizumab in locally recurrent and MBC patients demonstrated higher than anticipated toxicity while exhibiting only modest activity. Based on these results, we would not consider this combination for further investigation in this setting.