Cryopreserved human haematopoietic stem cells retain engraftment potential after extended (5-14 years) cryostorage

Harvesting of stem cells during the early phases of treatment with no immediate intention to perform a stem cell transplant is becoming an increasingly common practice. Such "insurance" harvests are often stored for many years before being needed for transplant in a subsequent relapse. The effect of long-term cryostorage (5-14 years) on the viability and functional capacity of haematopoietic stem cells (HSCs) was investigated in 40 bone marrow and peripheral blood harvests using standard in vitro methods, the colony forming unit-granulocyte/macrophage (CFU-GM) assay and a single platform viable CD34(+) cell absolute count by flow cytometry. Forty percent of harvests had CD34(+) HSC counts of at least 0.7 x 10(6)/kg bodyweight and 85% had CFU-GM counts of at least 1.0 x 10(5)/kg bodyweight, these values representing our institutional minimum requirements for safe transplantation. Based on these results, it appears that HSC collections can remain adequate for safe transplantation after up to 14 years of cryostorage. However, as deterioration of HSC quality and viability may occur, some precautions may be warranted, namely harvesting higher than normal numbers of HSCs in collections intended for long-term storage and repeating in vitro assays on harvests after long-term storage prior to transplantation.     

Preferential expression of a plant cystatin at nematode feeding sites confers resistance to Meloidogyne incognita and Globodera pallida

The expression patterns of three promoters preferentially active in the roots of Arabidopsis thaliana have been investigated in transgenic potato plants in response to plant parasitic nematode infection. Promoter regions from the three genes, TUB-1, ARSK1 and RPL16A were linked to the GUS reporter gene and histochemical staining was used to localize expression in potato roots in response to infection with both the potato cyst nematode, Globodera pallida and the root-knot nematode, Meloidogyne incognita. All three promoters directed GUS expression chiefly in root tissue and were strongly up-regulated in the galls induced by feeding M. incognita. Less activity was associated with the syncytial feeding cells of the cyst nematode, although the ARSK1 promoter was highly active in the syncytia of G. pallida infecting soil grown plants. Transgenic potato lines that expressed the cystatin OcIDeltaD86 under the control of the three promoters were evaluated for resistance against Globodera sp. in a field trial and against M. incognita in containment. Resistance to Globodera of 70 +/- 4% was achieved with the best line using the ARSK1 promoter with no associated yield penalty. The highest level of partial resistance achieved against M. incognita was 67 +/- 9% using the TUB-1 promoter. In both cases this was comparable to the level of resistance achieved using the constitutive cauliflower mosaic virus 35S (CaMV35S) promoter. The results establish the potential for limiting transgene expression in crop plants whilst maintaining efficacy of the nematode defence.     

The diheme cytochrome c peroxidase from Shewanella oneidensis requires reductive activation

We report the characterization of the diheme cytochrome c peroxidase (CcP) from Shewanella oneidensis (So) using UV-visible absorbance, electron paramagnetic resonance spectroscopy, and Michaelis-Menten kinetics. While sequence alignment with other bacterial diheme cytochrome c peroxidases suggests that So CcP may be active in the as-isolated state, we find that So CcP requires reductive activation for full activity, similar to the case for the canonical Pseudomonas type of bacterial CcP enzyme. Peroxide turnover initiated with oxidized So CcP shows a distinct lag phase, which we interpret as reductive activation in situ. A simple kinetic model is sufficient to recapitulate the lag-phase behavior of the progress curves and separate the contributions of reductive activation and peroxide turnover. The rates of catalysis and activation differ between MBP fusion and tag-free So CcP and also depend on the identity of the electron donor. Combined with Michaelis-Menten analysis, these data suggest that So CcP can accommodate electron donor binding in several possible orientations and that the presence of the MBP tag affects the availability of certain binding sites. To further investigate the structural basis of reductive activation in So CcP, we introduced mutations into two different regions of the protein that have been suggested to be important for reductive activation in homologous bacterial CcPs. Mutations in a flexible loop region neighboring the low-potential heme significantly increased the activation rate, confirming the importance of flexible loop regions of the protein in converting the inactive, as-isolated enzyme into the activated form.     

An fMRI Study Exploring the Overlap and Differences between Neural Representations of Physical and Recalled Pain

Implementing a recall paradigm without hypnosis, we use functional MRI (fMRI) to explore and compare nociceptive and centrally-driven pain experiences. We posit that a trace of a recent nociceptive event can be used to create sensory-re-experiencing of pain that can be qualified in terms of intensity and vividness. Fifteen healthy volunteers received three levels of thermal stimuli (warm, low pain and high pain) and subsequently were asked to recall and then rate this experience. Neuroimaging results reveal that recalling a previous sensory experience activates an extensive network of classical pain processing structures except the contralateral posterior insular cortex. Nociceptive-specific activation of this structure and the rated intensity difference between physical and recalled pain events allow us to investigate the link between the quality of the original nociceptive stimulus and the mental trace, as well as the differences between the accompanying neural responses. Additionally, by incorporating the behavioural ratings, we explored which brain regions were separately responsible for generating either an accurate or vivid recall of the physical experience. Together, these observations further our understanding of centrally-mediated pain experiences and pain memory as well as the potential relevance of these factors in the maintenance of chronic pain.     

Bisphenol A: developmental toxicity from early prenatal exposure

Bisphenol A (BPA) exposure has been documented in pregnant women, but consequences for development are not yet widely studied in human populations. This review presents research on the consequences for offspring of BPA exposure during pregnancy. Extensive work in laboratory rodents has evaluated survival and growth of the conceptus, interference with embryonic programs of development, morphological sex differentiation, sex differentiation of the brain and behavior, immune responsiveness, and mechanism of action. Sensitive measures include RAR, aryl hydrocarbon receptor, and Hox A10 gene expression, anogenital distance, sex differentiation of affective and exploratory behavior, and immune hyperresponsiveness. Many BPA effects are reported at low doses (10–50 µg/kg d range) by the oral route of administration. At high doses (>500,000 µg/kg d) fetal viability is compromised. Much of the work has centered around the implications of the estrogenic actions of this agent. Some work related to thyroid mechanism of action has also been explored. BPA research has actively integrated current knowledge of developmental biology, concepts of endocrine disruption, and toxicological research to provide a basis for human health risk assessment. Birth Defects Res (Part B) 89:441–466, 2010. © 2010 Wiley‐Liss, Inc.     

Primary Succession on a Hawaiian Dryland Chronosequence

We used measurements from airborne imaging spectroscopy and LiDAR to quantify the biophysical structure and composition of vegetation on a dryland substrate age gradient in Hawaii. Both vertical stature and species composition changed during primary succession, and reveal a progressive increase in vertical stature on younger substrates followed by a collapse on Pleistocene-aged flows. Tall-stature Metrosideros polymorpha woodlands dominated on the youngest substrates (hundreds of years), and were replaced by the tall-stature endemic tree species Myoporum sandwicense and Sophora chrysophylla on intermediate-aged flows (thousands of years). The oldest substrates (tens of thousands of years) were dominated by the short-stature native shrub Dodonaea viscosa and endemic grass Eragrostis atropioides. We excavated 18 macroscopic charcoal fragments from Pleistocene-aged substrates. Mean radiocarbon age was 2,002 years and ranged from < 200 to 7,730. Genus identities from four fragments indicate that Osteomeles spp. or M. polymorpha once occupied the Pleistocene-aged substrates, but neither of these species is found there today. These findings indicate the existence of fires before humans are known to have occupied the Hawaiian archipelago, and demonstrate that a collapse in vertical stature is prevalent on the oldest substrates. This work contributes to our understanding of prehistoric fires in shaping the trajectory of primary succession in Hawaiian drylands.     

Actin redistribution in mosquito malpighian tubules after a blood meal and cyclic AMP stimulation

Fluid secretion by mosquito Malpighian tubules is critical to maintaining fluid and electrolyte balance after a blood meal. Endogenous cAMP levels increase in Malpighian tubules after a blood meal. Here, we determined if corresponding changes in intracellular actin distribution occur after a blood meal or dibutyryl-cAMP (db-cAMP) stimulation and whether altering actin turnover inhibits secretion. In untreated Malpighian tubules, beta-actin immunostaining was more intense in the apical region of adult Malpighian tubules than in the cytoplasm. Stimulation by a blood meal or db-cAMP significantly decreased beta-actin immunostaining in the non-apical region of the cell. Db-cAMP had similar effects in larvae and pupae Malpighian tubules. In contrast, no detectable shift in F-actin distribution was detected; however, F-actin bundles within the cytoplasm increased in size after treatment with db-cAMP. Pretreatment of Malpighian tubules with agents perturbing actin fiber assembly and disassembly decreased basal secretion rates and inhibited the stimulatory effects of db-cAMP. Our results show (1) beta-actin redistributes toward the apical membrane after a blood meal and this correlates temporally with increase urine flow rate and intracellular cAMP levels, (2) Malpighian tubules from all developmental stages exhibit this same response to db-cAMP-stimulation, and (3) dynamic assembly and disassembly of beta-actin is required for db-cAMP-stimulated secretion.