Transmitted/Founder Simian Immunodeficiency Virus Envelope Sequences in Vesicular Stomatitis and Semliki Forest Virus Vector Immunized Rhesus Macaques

Identification of transmitted/founder simian immunodeficiency virus (SIV) envelope sequences responsible for infection may prove critical for understanding HIV/SIV mucosal transmission. We used single genome amplification and phylogenetic analyses to characterize transmitted/founder SIVs both in the inoculum and in immunized-infected rhesus monkeys. Single genome amplification of the SIVsmE660 inoculum revealed a maximum diversity of 1.4%. We also noted that the consensus sequence of the challenge stock differed from the vaccine construct in 10 amino acids including 3 changes in the V4 loop. Viral env was prepared from rhesus plasma in 3 groups of 6 immunized with vesicular stomatitis virus (VSV) vectors and boosted with Semliki forest virus (SFV) replicons expressing (a) SIVsmE660 gag-env (b) SIVsmE660 gag-env plus rhesus GM-CSF and (c) control influenza hemagglutinin protein. Macaques were immunized twice with VSV-vectors and once with SFV vector and challenged intrarectally with 4000 TCID₅₀. Single genome amplification characterized the infections of 2 unprotected animals in the gag-env immunized group, both of which had reduced acute plasma viral loads that ended as transient infections indicating partial immune control. Four of 6 rhesus were infected in the gag-env + GM-CSF group which demonstrated that GM-CSF abrogated protection. All 6 animals from the control group were infected having high plasma viral loads. We obtained 246 full-length envelope sequences from SIVsmE660 infected macaques at the peak of infection and determined the number of transmitted/founder variants per animal. Our analysis found that 2 of 2 gag-env vaccinated but infected macaques exhibited single but distinct virus envelope lineages whereas rhesus vaccinated with gag-env-GM-CSF or HA control exhibited both single and multiple env lineages. Because there were only 2 infected animals in the gag-env vaccinated rhesus compared to 10 infected rhesus in the other 2 groups, the significance of finding single env variants in the gag-env vaccinated group could not be established.     

Decrease in Vitamin D Status in the Greenlandic Adult Population from 1987–2010

Background

Low vitamin D status may be pronounced in Arctic populations due to limited sun exposure and decreasing intake of traditional food.

Objective

To investigate serum 25(OH)D3 as a measure of vitamin D status among adult Inuit in Greenland, predictors of low serum 25(OH)D3 concentrations and the trend from 1987 to 2005–2010.

Design

A total of 2877 randomly selected Inuit (≥18 years) from the Inuit Health in Transition study were included. A sub-sample (n = 330) donated a blood sample in 1987 which allowed assessment of time trends in vitamin D status.

Results

The geometric mean serum 25(OH)D3 (25[OH]D2 concentrations were negligible and not reported) in 2005–2010 was lowest among the 18–29 year old individuals (30.7 nmol/L; 95% CI: 29.7; 31.7) and increased with age. In all age-groups it decreased from 1987 to 2005–2010 (32%–58%). Low 25(OH)D3 concentrations (<50 nmol/L) were present in 77% of the 18–29 year old and decreased with age. A characteristic seasonal variation in 25(OH)D3 concentrations was observed (range 33.2–57.1 nmol/L, p<0.001), with the highest concentrations in August to October. Age (2.0% per year increase; CI: 1.7, 2.2), female gender (7.1%; CI: 2.0; 12.5), alcohol intake (0.2% per increase in drinks/week; 0.0; 0.4), and traditional diet (10.0% per 100 g/d increase; CI: 7.9; 12.1) were associated with increased serum 25(OH)D3, whereas smoking (−11.6%; CI: −16.2; −6.9), BMI (−0.6%; CI: −1.1; −0.2) and latitude (−0.7% per degree increase; CI: −1.3; −0.2) were associated with decreased concentrations.

Conclusion

We identified a remarkable decrease in vitamin D status from 1987 to 2005–2010 and a presently low vitamin D status among Inuit in Greenland. A change away from a traditional diet may well explain the observed decline. The study argues for the need of increased dietary intake of vitamin D and supplementation might be considered.     

Characterization of Discrete Subpopulations of Progenitor Cells in Traumatic Human Extremity Wounds

Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds, each having the ability to differentiate into multiple cells types in vitro. A crude cell suspension derived from traumatized muscle was positively sorted for CD29, CD31, CD34, CD56 or CD91. The cell suspension was also simultaneously negatively sorted for either CD45 or CD117 to exclude hematopoietic stem cells. These subpopulations varied in terms their total numbers and their abilities to grow, migrate, differentiate and secrete cytokines. While all five subpopulations demonstrated equal abilities to undergo osteogenesis, they were distinct in their ability to undergo adipogenesis and vascular endotheliogenesis. The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly. The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2. When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells. These data indicate that while there are multiple cell types within traumatized muscle that have osteogenic differentiation capacity and may contribute to bone formation in post-traumatic heterotopic ossification (HO), the major contributory cell types are CD29+ and CD34+, which demonstrate endothelial progenitor cell characteristics.     

Structural basis for the light regulation of chloroplast NADP malate dehydrogenase

The activity of chloroplast NADP-malate dehydrogenase (NADP-MDH; EC 1.1.1.82) in both C3 and C4 plants is regulated by light intensity. In darkness, the activity of the enzyme can be less than 1% of the maximal activity found at high light intensities. The extent of activation in the light is dynamic, responding rapidly to changes in light intensity and adapting to changes in photosynthetic rate. Enzyme activation is caused by thioredoxin-catalyzed reduction of two regulatory disulfide bonds, while inactivation is accomplished by thioredoxin-catalyzed re-oxidation. In the case of NADP-MDH, the coenzyme substrates NADP+ and NADPH modify the rate of this interconversion and seem to be important to the extent of activation in vivo. The recent determination of the X-ray structure of the oxidized, dark form of NADP-MDH from the C4 plants Flaveria bidentis and Sorghum shows how oxidation of a disulfide bond can inactivate the enzyme. This review discusses the various structural features of NADP-MDH that seem to be responsible for the regulatory properties of the enzyme and emphasizes that large changes of activity can be accomplished by multiple, small, reinforcing changes rather than a single large change in a signal molecule concentration.     

Real-time and Label-free Bio-sensing of Molecular Interactions by Surface Plasmon Resonance: A Laboratory Medicine Perspective

Radioactive, chromogenic, fluorescent and other labels have long provided the basis of detection systems for biomolecular interactions including immunoassays and receptor binding studies. However there has been unprecedented growth in a number of powerful label free biosensor technologies over the last decade. While largely at the proof-of-concept stage in terms of clinical applications, the development of more accessible platforms may see surface plasmon resonance (SPR) emerge as one of the most powerful optical detection platforms for the real-time monitoring of biomolecular interactions in a label-free environment.In this review, we provide an overview of SPR principles and current and future capabilities in a diagnostic context, including its application for monitoring a wide range of molecular markers of disease. The advantages and pitfalls of using SPR to study biomolecular interactions are discussed, with particular emphasis on its potential to differentiate subspecies of analytes and the inherent ability for quantitation through calibration-free concentration analysis (CFCA). In addition, recent advances in multiplex applications, high throughput arrays, miniaturisation, and enhancements using noble metal nanoparticles that promise unprecedented sensitivity to the level of single molecule detection, are discussed.In summary, while SPR is not a new technique, technological advances may see SPR quickly emerge as a highly powerful technology, enabling rapid and routine analysis of molecular interactions for a diverse range of targets, including those with clinical applicability. As the technology produces data quickly, in real-time and in a label-free environment, it may well have a significant presence in future developments in lab-on-a-chip technologies including point-of-care devices and personalised medicine.     

Auditory Discrimination Learning: Role of Working Memory

Perceptual training is generally assumed to improve perception by modifying the encoding or decoding of sensory information. However, this assumption is incompatible with recent demonstrations that transfer of learning can be enhanced by across-trial variation of training stimuli or task. Here we present three lines of evidence from healthy adults in support of the idea that the enhanced transfer of auditory discrimination learning is mediated by working memory (WM). First, the ability to discriminate small differences in tone frequency or duration was correlated with WM measured with a tone n-back task. Second, training frequency discrimination around a variable frequency transferred to and from WM learning, but training around a fixed frequency did not. The transfer of learning in both directions was correlated with a reduction of the influence of stimulus variation in the discrimination task, linking WM and its improvement to across-trial stimulus interaction in auditory discrimination. Third, while WM training transferred broadly to other WM and auditory discrimination tasks, variable-frequency training on duration discrimination did not improve WM, indicating that stimulus variation challenges and trains WM only if the task demands stimulus updating in the varied dimension. The results provide empirical evidence as well as a theoretic framework for interactions between cognitive and sensory plasticity during perceptual experience.