The first cytoplasmic loop of the mannitol permease from Escherichia coli is accessible for sulfhydryl reagents from the periplasmic side of the membrane

The mannitol permease (EII(Mtl)) from Escherichia coli couples mannitol transport to phosphorylation of the substrate. Renewed topology prediction of the membrane-embedded C domain suggested that EII(Mtl) contains more membrane-embedded segments than the six proposed previously on the basis of a PhoA fusion study. Cysteine accessibility was used to confirm this notion. Since cysteine 384 in the cytoplasmic B domain is crucial for the phosphorylation activity of EII(Mtl), all cysteine mutants contained this activity-linked cysteine residue in addition to those introduced for probing the membrane topology of the protein. To distinguish between the activity-linked cysteine and the probed cysteine, either trypsin was used to specifically digest the two cytoplasmic domains (A and B), thereby removing Cys384, or Cys384 was protected by phosphorylation from alkylation by N-ethylmaleimide (NEM). Our data show that upon phosphorylation EII(Mtl) undergoes major conformational changes, whereby residues in the putative first cytoplasmic loop become accessible to NEM. Other residues in this loop were accessible to NEM in intact cells and inside-out membrane vesicles, but cysteine residues at these positions only reacted with the membrane-impermeable sulfhydryl reagent from the periplasmic side of the protein. These and other results suggest that the predicted loop between TM2 and TM3 may fold back into the membrane and form part of the translocation path.     

Magnetic resonance tracking of dendritic cells in melanoma patients for monitoring of cellular therapy

The success of cellular therapies will depend in part on accurate delivery of cells to target organs. In dendritic cell therapy, in particular, delivery and subsequent migration of cells to regional lymph nodes is essential for effective stimulation of the immune system. We show here that in vivo magnetic resonance tracking of magnetically labeled cells is feasible in humans for detecting very low numbers of dendritic cells in conjunction with detailed anatomical information. Autologous dendritic cells were labeled with a clinical superparamagnetic iron oxide formulation or (111)In-oxine and were co-injected intranodally in melanoma patients under ultrasound guidance. In contrast to scintigraphic imaging, magnetic resonance imaging (MRI) allowed assessment of the accuracy of dendritic cell delivery and of inter- and intra-nodal cell migration patterns. MRI cell tracking using iron oxides appears clinically safe and well suited to monitor cellular therapy in humans.     

Enhancing scatterplots with smoothed densities

MOTIVATION: Scatterplots of microarray data generally contain a very large number of dots, making it difficult to get a good impression of their distribution in dense areas. RESULTS: We present a fast and simple algorithm for two-dimensional histogram smoothing, to visually enhance scatterplots. AVAILABILITY: Functions for Matlab and R are available from the corresponding author.     

Diurnal Patterns and Correlates of Older Adults’ Sedentary Behavior

Introduction

Insights into the diurnal patterns of sedentary behavior and the identification of subgroups that are at increased risk for engaging in high levels of sedentary behavior are needed to inform potential interventions for reducing older adults’ sedentary time. Therefore, we examined the diurnal patterns and sociodemographic correlates of older adults’ sedentary behavior(s).

Methods

Stratified cluster sampling was used to recruit 508 non-institutionalized Belgian older adults (≥ 65 years). Morning, afternoon, evening and total sedentary time was assessed objectively using accelerometers. Specific sedentary behaviors, total sitting time and sociodemographic attributes were assessed using an interviewer-administered questionnaire.

Results

Participants self-reported a median of 475 (Q1-Q3 = 383–599) minutes/day of total sitting time and they accumulated a mean of 580 ± 98 minutes/day of accelerometer-derived sedentary time. Sedentary time was lowest during the morning and highest during the evening. Older participants were as sedentary as younger participants during the evening, but they were more sedentary during daytime. Compared to married participants, widowers were more sedentary during daytime. Younger participants (< 75 years), men and the higher educated were more likely to engage in (high levels of) sitting while driving a car and using the computer. Those with tertiary education viewed 29% and 22% minutes/day less television compared to those with primary or secondary education, respectively. Older participants accumulated 35 sedentary minutes/day more than did younger participants and men accumulated 32 sedentary minutes/day more than did women.

Conclusion

These findings highlight diurnal variations and potential opportunities to tailor approaches to reducing sedentary time for subgroups of the older adult population.     

Telemedicine’s Potential to Support Good Dying in Nigeria: A Qualitative Study

Objectives

This qualitative study explores Nigerian health care professionals’ concepts of good dying/a good death and how telemedicine technologies and services would fit the current Nigerian palliative care practice.

Materials and Methods

Supported by the Centre for Palliative Care Nigeria (CPCN) and the University College Hospital (UCH) in Ibadan, Nigeria, the authors organized three focus groups with Nigerian health care professionals interested in palliative care, unstructured interviews with key role players for palliative care and representatives of telecom companies, and field visits to primary, secondary and tertiary healthcare clinics that provided palliative care. Data analysis consisted of open coding, constant comparison, diagramming of categorizations and relations, and extensive member checks.

Results

The focus group participants classified good dying into 2 domains: a feeling of completion of the individual life and dying within the community. Reported barriers to palliative care provision were socio-economic consequences of being seriously ill, taboos on dying and being ill, restricted access to adequate medical–technical care, equation of religion with medicine, and the faulty implementation of palliative care policy by government. The addition of telemedicine to Nigeria’s palliative care practice appears problematic, due to irregular bandwidth, poor network coverage, and unstable power supply obstructing interactivity and access to information. However, a tele-education ‘lite’ scenario seemed viable in Nigeria, wherein low-tech educational networks are central that build on non-synchronous online communication.

Discussion

Nigerian health care professionals’ concepts on good dying/a good death and barriers and opportunities for palliative care provision were, for the greater part, similar to prior findings from other studies in Africa. Information for and education of patient, family, and community are essential to further improve palliative care in Africa. Telemedicine can only help if low-tech solutions are applied that work around network coverage problems by focusing on non-synchronous online communication.     

The impact of slow stomatal kinetics on photosynthesis and water use efficiency under fluctuating light

Dynamic light conditions require continuous adjustments of stomatal aperture. The kinetics of stomatal conductance (g_s) is hypothesized to be key to plant productivity and water use efficiency (WUE). Using step-changes in light intensity, we studied the diversity of light-induced g_s kinetics in relation to stomatal anatomy in five banana genotypes (Musa spp.) and modeled the impact of both diffusional and biochemical limitations on photosynthesis (A). The dominant A limiting factor was the diffusional limitation associated with g_s kinetics. All genotypes exhibited a strong limitation of A by g_s, indicating a priority for water saving. Moreover, significant genotypic differences in g_s kinetics and g_s limitations of A were observed. For two contrasting genotypes, the impact of differential g_s kinetics was further investigated under realistic diurnally fluctuating light conditions and at the whole-plant level. Genotype-specific stomatal kinetics observed at the leaf level was corroborated at whole-plant level by transpiration dynamics, validating that genotype-specific responses are still maintained despite differences in g_s control at different locations in the leaf and across leaves. However, under diurnally fluctuating light conditions the impact of g_s speediness on A and intrinsic (_iWUE) depended on time of day. During the afternoon there was a setback in kinetics: absolute g_s and g_s responses to light were damped, strongly limiting A and impacting diurnal _iWUE. We conclude the impact of differential g_s kinetics depended on target light intensity, magnitude of change, g_s prior to the change in light intensity, and particularly time of day.     

Origins of neocortical interneurons in mice

GABAergic interneurons influence the development and function of the cerebral cortex through the actions of a variety of subtypes. Despite the relevance to cortical function and dysfunction, including seizure disorders and neuropsychiatric illnesses, the molecular determinants of interneuron fate remain largely unidentified. Challenges to this endeavor include the difficulty of studying fate determination of cells that even in rodents do not fully mature until weeks after their embryonic birth. However, in recent years a strong literature has grown on the temporal and spatial origins of distinct interneuron groups and types. Here we seek to highlight these findings, particularly in mice. Our goal is to lay the groundwork for future studies that use mouse genetics to study cortical interneuron fate determination and function.