NPC1 regulates ER contacts with endocytic organelles to mediate cholesterol egress.

Transport of dietary cholesterol from endocytic organelles to the endoplasmic reticulum (ER) is essential for cholesterol homoeostasis, but the mechanism and regulation of this transport remains poorly defined. Membrane contact sites (MCS), microdomains of close membrane apposition, are gaining attention as important platforms for non-vesicular, inter-organellar communication. Here we investigate the impact of ER-endocytic organelle MCS on cholesterol transport. We report a role for Niemann-Pick type C protein 1 (NPC1) in tethering ER-endocytic organelle MCS where it interacts with the ER-localised sterol transport protein Gramd1b to regulate cholesterol egress. We show that artificially tethering MCS rescues the cholesterol accumulation that characterises NPC1-deficient cells, consistent with direct lysosome to ER cholesterol transport across MCS. Finally, we identify an expanded population of lysosome-mitochondria MCS in cells depleted of NPC1 or Gramd1b that is dependent on the late endosomal sterol-binding protein STARD3, likely underlying the mitochondrial cholesterol accumulation in NPC1-deficient cells.

Correction of H3+ contributions in hydrogen isotope ratio monitoring mass spectrometry.

Two fundamentally different approaches, termed "pointwise" and "peakwise," are currently used to correct hydrogen isotope ratio monitoring data for the presence of H3+ ion contributions. Consideration of the underlying assumptions shows that the peakwise approach is valid only for peaks with the same functional shape and only when background signals do not vary. The pointwise correction is much more versatile and can be used even when peak shapes and sizes, as well as background signals, vary significantly. It is not exact and is limited in accuracy by (1) the signal-broadening effects of electronic time constants, (2) the analog-to-digital conversion frequency, and (3) the highest frequency of the sample signal. To minimize errors for typical gas chromatographic signals, time constants of <500 ms and analog-to-digital sampling intervals of < or =250 ms are needed. Errors are further minimized by matching sample and standard peaks in both amplitude and D/H ratio. Using the pointwise algorithm, we demonstrate that a series of 14 homologous n-alkanes varying in concentration over a 5-fold range can be analyzed with a mean precision of 2.3 per thousand and no systematic errors.

Determination of the the H3 factor in hydrogen isotope ratio monitoring mass spectrometry.

The H3 factor, K, is a parameter required in high-precision, mass spectrometric analyses of hydrogen isotopic abundances. When H2 is used as the sample gas, R* = R - Ki2, where R* is the true HD/H2 ratio, R is the observed (mass 3)/(mass 2) ion-current ratio, and i2 is the ion current at mass 2. Four different methods for the determination of K were defined and tested under conditions characteristic of isotope ratio monitoring systems. Three of these were peak-based. The fourth employed steady flows of H2 from a conventional inlet system. Results obtained using the latter method were more precise (standard deviation of K = 0.1 versus approximately 0.6 ppm mV(-1) for the peak-based methods). However, use of the resulting values of K for correction of isotope ratio monitoring GC/MS results led to systematic errors as large as 9 per thousand, whereas use of the peak-based values led to no systematic errors. Values of K were only weakly dependent on the pressure of He, declining approximately 5% for each 10-fold increase in P(He). Small variations in partial pressures of H2O and CH4, potential contaminants under isotope ratio monitoring conditions, had no significant effect on values of K.

Space charge evaluation in a plasma-source mass spectrograph.

Space charge effects, and the matrix interferences they cause, are problems in inductively coupled plasma mass spectrometry (ICPMS). It has previously been observed that these deleterious space charge effects are not significantly present in sector-field instruments, a fact that has been attributed, but not demonstrated, to the high accelerating potentials they commonly employ. To examine the significance of space charge in our plasma-source mass spectrograph (which operates at only moderate accelerating potentials) and in other sector instruments, a graphite disk was placed approximately 7 cm behind the skimmer. An inductively coupled plasma was operated for 17 h while a 0.01 mM multielement solution was introduced. This disk was then analyzed by spatially resolved laser ablation ICP time-of-flight MS. Second vacuum-stage acceleration appears to be an important factor that governs the elemental distribution within the ion beam. The ion beam width at m/z 208 is one-third of its width at m/z 7 using an accelerating potential of 800 V; at an accelerating potential of 4000 V, the ion beam width does not vary with mass.

An introduction to ion optics for the mass spectrograph.

A mass spectrograph is an instrument that separates and simultaneously focuses ions, along a focal plane, of different mass/charge ratios that are diverging in direction and that have a variable velocity. With these instruments and a spatially sensitive ion detector, simultaneous detection can be employed, which has been shown to improve precision and throughput (as compared to a mass spectrometer that can only detect one mass at a time). Knowing how an ion beam focuses throughout the mass spectrograph and onto the focal plane is crucial. We present here rudimentary ion optics of the mass spectrograph in a simple yet useable manner. From there, we investigate the direction and energy focal lines of some mass-spectrograph geometries, using the ion optics presented. Lastly, other mass spectrograph geometries that fall outside the field of knowledge of the ion optics covered are discussed. With this review, we hope to provide an understandable and universal ion optic theory that encompasses a wide range of mass spectrographs and that is palatable to the novice as well as the expert. © 1997 John Wiley & Sons, Inc.

Comparison of the overall quality of life in 50 long-term survivors of autologous and allogeneic bone marrow transplantation.

Fifty long-term survivors of bone marrow transplant (mean post-transplant time = 42.4 months) participated in a study examining their psychosocial adjustment and quality of life. Differences between patients who received an autologous marrow transplant and those who received an allogeneic marrow transplant were identified. Patients with an autologous transplant had mainly psychological difficulties in their post-transplant adaptation, whereas patients with allogeneic transplant developed more physical problems. Overall, their psychosocial adjustment was similar and comparable with other medical groups of patients. A quarter of both groups had failed to return to work/education and up to 9.5% had difficulty in carrying out daily tasks. Twenty per cent of the patients with autologous transplant had clinical signs of anxiety and 10% clinical signs of depression, whereas there was an incidence of 10% of patients with allogeneic transplant with anxiety, but no cases with clinical depression. Family relationships were found to be more integrated and lower in conflict compared with normal families. Quality of life has been described as good to excellent in most of the patients. Multiple regression analysis showed that physical symptomatology, vocational adjustment and depression are predictors of the degree of the patients' quality of life.

A wind of change for entrenched behaviour. Getting the nursing process to work in ICU.

As the emphasis on individualised patient care increased in a large ICU, it was decided to apply the nursing process. This article describes how the process was adapted to suit the unit's special needs.