bluuebunny/crossref_metadata_2025 huggingface dataset:
The paradox of improved antiretroviral therapy in HIV: potential for nutritional modulation?
Authors: Ware Lisa J. , Wootton S. A.
Published: September 2008

Chronic infection with HIV type 1 is associated with alterations in macronutrient metabolism, specifically elevated plasma lipids, glucose and reduced insulin sensitivity. These alterations are most severe in patients at the later stages of AIDS, indicating a relationship with disease progression. Recently, a metabolic syndrome, termed lipodystrophy, has been described in successfully-treated HIV patients in whom the altered macronutrient metabolism of HIV infection appears to be amplified markedly, with concurrent alterations in adipose tissue patterning. This syndrome presents a paradox, as before the development of highly-active antiretroviral therapy (HAART) the most severe perturbations in metabolism were observed in the sickest patients. Now, the patients that respond well to therapy are showing metabolic perturbations much greater than those seen before. The implications of this syndrome are that, whilst life expectancy may be increased by reducing viral load, there are concomitant increases in the risk of cardiovascular disease, diabetes and pancreatitis within this patient population. The aetiology of the syndrome remains unclear. In a collaborative trial with the Chelsea and Westminster Hospital in London we have used stable-isotope-labelled fatty acids to examine the hypothesis that treatment with HAART causes a delayed clearance of dietary lipid from the circulation, resulting in the retention of lipid within plasma and the downstream changes in insulin and glucose homeostasis. This hypothesis would indicate a role for low-fat diets, exercise and drugs that reduce plasma lipid or insulin resistance, in modulating the response to antiretroviral therapy in HIV infection.
Nutritional losses and gains during processing: future problems and issues†
Authors: Henry* C. J. K. , Heppell N.
Published: September 2008

Developments in food technology and nutrition today are shaping our food supply in an unprecedented way. They have not only helped produce a variety of foods with varying taste and texture but also optimally retain the nutritional quality of food. Increased use of novel ingredients in the future manufacture of foods is likely to reduce the amount of micronutrients available in our diet. To combat this, food fortification may be more widely used. Collaboration between food technologists and nutritionists is imperative if we are to see the continued manufacture of wholesome food.
Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ
Authors: Trayhurn Paul , Beattie John H.
Published: September 2008

The traditional role attributed to white adipose tissue is energy storage, fatty acids being released when fuel is required. The metabolic role of white fat is, however, complex. For example, the tissue is needed for normal glucose homeostasis and a role in inflammatory processes has been proposed. A radical change in perspective followed the discovery of leptin; this critical hormone in energy balance is produced principally by white fat, giving the tissue an endocrine function. Leptin is one of a number of proteins secreted from white adipocytes, which include angiotensinogen, adipsin, acylation-stimulating protein, adiponectin, retinol-binding protein, tumour neorosis factor a, interleukin 6, plasminogen activator inhibitor-1 and tissue factor. Some of these proteins are inflammatory cytokines, some play a role in lipid metabolism, while others are involved in vascular haemostasis or the complement system. The effects of specific proteins may be autocrine or paracrine, or the site of action may be distant from adipose tissue. The most recently described adipocyte secretory proteins are fasting-induced adipose factor, a fibrinogen–angiopoietin-related protein, metallothionein and resistin. Resistin is an adipose tissue-specific factor which is reported to induce insulin resistance, linking diabetes to obesity. Metallothionein is a metal-binding and stress-response protein which may have an antioxidant role. The key challenges in establishing the secretory functions of white fat are to identify the complement of secreted proteins, to establish the role of each secreted protein, and to assess the pathophysiological consequences of changes in adipocyte protein production with alterations in adiposity (obesity, fasting, cachexia). There is already considerable evidence of links between increased production of some adipocyte factors and the metabolic and cardiovascular complications of obesity. In essence, white adipose tissue is a major secretory and endocrine organ involved in a range of functions beyond simple fat storage.
Aftiphilin and γ-Synergin Are Required for Secretagogue Sensitivity of Weibel-Palade Bodies in Endothelial Cells
Authors: Lui-Roberts Winnie W.Y. , Ferraro Francesco , Nightingale Thomas D. , Cutler Daniel F.
Published: September 2008

Formation of secretory organelles requires the coupling of cargo selection to targeting into the correct exocytic pathway. Although the assembly of regulated secretory granules is driven in part by selective aggregation and retention of content, we recently reported that adaptor protein-1 (AP-1) recruitment of clathrin is essential to the initial formation of Weibel-Palade bodies (WPBs) at the trans-Golgi network. A selective co-aggregation process might include recruitment of components required for targeting to the regulated secretory pathway. However, we find that acquisition of the regulated secretory phenotype by WPBs in endothelial cells is coupled to but can be separated from formation of the distinctive granule core by ablation of the AP-1 effectors aftiphilin and γ-synergin. Their depletion by small interfering RNA leads to WPBs that fail to respond to secretagogue and release their content in an unregulated manner. We find that these non-responsive WPBs have density, markers of maturation, and highly multimerized von Willebrand factor similar to those of wild-type granules. Thus, by also recruiting aftiphilin/γ-synergin in addition to clathrin, AP-1 coordinates formation of WPBs with their acquisition of a regulated secretory phenotype.
INF1 Is a Novel Microtubule-associated Formin
Authors: Young Kevin G. , Thurston Susan F. , Copeland Sarah , Smallwood Chelsea , Copeland John W.
Published: September 2008

Formin proteins, characterized by the presence of conserved formin homology (FH) domains, play important roles in cytoskeletal regulation via their abilities to nucleate actin filament formation and to interact with multiple other proteins involved in cytoskeletal regulation. The C-terminal FH2 domain of formins is key for actin filament interactions and has been implicated in playing a role in interactions with microtubules. Inverted formin 1 (INF1) is unusual among the formin family in having the conserved FH1 and FH2 domains in its N-terminal half, with its C-terminal half being composed of a unique polypeptide sequence. In this study, we have examined a potential role for INF1 in regulating microtubule structure. INF1 associates discretely with microtubules, and this association is dependent on a novel C-terminal microtubule-binding domain. INF1 expressed in fibroblast cells induced actin stress fiber formation, coalignment of microtubules with actin filaments, and the formation of bundled, acetylated microtubules. Endogenous INF1 showed an association with acetylated microtubules, and knockdown of INF1 resulted in decreased levels of acetylated microtubules. Our data suggests a role for INF1 in microtubule modification and potentially in coordinating microtubule and F-actin structure.
Glycosylated SV2A and SV2B Mediate the Entry of Botulinum Neurotoxin E into Neurons
Authors: Dong Min , Liu Huisheng , Tepp William H. , Johnson Eric A. , Janz Roger , Chapman Edwin R.
Published: September 2008

Botulinum neurotoxin E (BoNT/E) can cause paralysis in humans and animals by blocking neurotransmitter release from presynaptic nerve terminals. How this toxin targets and enters neurons is not known. Here we identified two isoforms of the synaptic vesicle protein SV2, SV2A and SV2B, as the protein receptors for BoNT/E. BoNT/E failed to enter neurons cultured from SV2A/B knockout mice; entry was restored by expressing SV2A or SV2B, but not SV2C. Mice lacking SV2B displayed reduced sensitivity to BoNT/E. The fourth luminal domain of SV2A or SV2B alone, expressed in chimeric receptors by replacing the extracellular domain of the low-density lipoprotein receptor, can restore the binding and entry of BoNT/E into neurons lacking SV2A/B. Furthermore, we found disruption of a N-glycosylation site (N573Q) within the fourth luminal domain of SV2A rendered the mutant unable to mediate the entry of BoNT/E and also reduced the entry of BoNT/A. Finally, we demonstrate that BoNT/E failed to bind and enter ganglioside-deficient neurons; entry was rescued by loading exogenous gangliosides into neuronal membranes. Together, the data reported here demonstrate that glycosylated SV2A and SV2B act in conjunction with gangliosides to mediate the entry of BoNT/E into neurons.
A new rule-based algorithm for identifying metabolic markers in prostate cancer using tandem mass spectrometry
Authors: Osl Melanie , Dreiseitl Stephan , Pfeifer Bernhard , Weinberger Klaus , Klocker Helmut , Bartsch Georg , Schäfer Georg , Tilg Bernhard , Graber Armin , Baumgartner Christian
Published: September 2008

Motivation: Prostate cancer is the most prevalent tumor in males and its incidence is expected to increase as the population ages. Prostate cancer is treatable by excision if detected at an early enough stage. The challenges of early diagnosis require the discovery of novel biomarkers and tools for prostate cancer management.

Results: We developed a novel feature selection algorithm termed as associative voting (AV) for identifying biomarker candidates in prostate cancer data measured via targeted metabolite profiling MS/MS analysis. We benchmarked our algorithm against two standard entropy-based and correlation-based feature selection methods [Information Gain (IG) and ReliefF (RF)] and observed that, on a variety of classification tasks in prostate cancer diagnosis, our algorithm identified subsets of biomarker candidates that are both smaller and show higher discriminatory power than the subsets identified by IG and RF. A literature study confirms that the highest ranked biomarker candidates identified by AV have independently been identified as important factors in prostate cancer development.

Availability: The algorithm can be downloaded from the following http://biomed.umit.at/page.cfm?pageid=516

Contact:  melanie.osl@umit.at
Environmental indicators: their utility in meeting the OSPAR Convention's regulatory needs
Authors: Johnson David
Published: September 2008

Johnson, D. 2008. Environmental indicators: their utility in meeting the OSPAR Convention's regulatory needs. – ICES Journal of Marine Science, 65: 1387–1391. The role of marine environmental indicators is clear; selecting indicators to inform management and for the development of accepted frameworks is proving more elusive. The OSPAR Commission, charged with the environmental protection of the Northeast Atlantic, has been applying an ecosystem approach since 1998, applying on a trial basis a system of ecological quality objectives as indicators of ecosystem health for the North Sea. This paper presents a framework for biodiversity monitoring and assessment, and discusses specific issues associated with marine-litter indicators, representing a cell within the matrix of the framework. In the immediate future, European marine legislation will drive the development of environmental indicators in the area, and further harmonization of indicator sets should enhance their utility.
Monitoring Mortality of Pythium Zoospores in Chlorinated Water Using Oxidation Reduction Potential
Authors: Lang Jillian M. , Rebits Brittany , Newman Steven E. , Tisserat Ned
Published: September 2008

Pythium species are frequently recovered from recycled irrigation water in greenhouse production systems and may cause damping off and root rot if the water is not disinfested properly. Chlorination is often employed as a disinfesting agent, but can be difficult to monitor accurately because its activity is pH-dependent. Oxidation reduction potential (ORP) is a reliable, real-time measurement of the oxidizing potential of a chlorine solution. We exposed zoospores of Pythium aphanidermatum and P. dissotocum municipal water where the ORP was increased by adding 0.125, 0.5, and 2 mg/liter chlorine (pH 7.6 to 8.1) or to the same water source where the pH was lowered to 6.0 prior to the addition of chlorine, resulting in a final pH of 6 to 7.3. Some zoospores of P. aphanidermatum and P. dissotocum survived treatment to the highest chlorine concentration for 4 min in water where pH was not lowered. When the water pH was lowered to 6.0 prior to chlorine addition, 100% of the P. aphanidermatum zoospores were killed after 0.5 min exposure to 0.5 mg/liter chlorine, where ORP ranged from 748 to 790 mV and pH 6.3. Lowering the initial water pH improved disinfestation of P. dissotocum zoospores at the highest chlorine concentration tested and a mean ORP of 790 mV.

Accepted for publication 7 July 2008. Published 22 September 2008.
Molecular Characterization of Organelle-Type Nudix Hydrolases in Arabidopsis
Authors: Ogawa Takahisa , Yoshimura Kazuya , Miyake Hiroe , Ishikawa Kazuya , Ito Daisuke , Tanabe Noriaki , Shigeoka Shigeru
Published: September 2008

Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases act to hydrolyze ribonucleoside and deoxyribonucleoside triphosphates, nucleotide sugars, coenzymes, or dinucleoside polyphosphates. Arabidopsis (Arabidopsis thaliana) contains 27 genes encoding Nudix hydrolase homologues (AtNUDX1 to -27) with a predicted distribution in the cytosol, mitochondria, and chloroplasts. Previously, cytosolic Nudix hydrolases (AtNUDX1 to -11 and -25) were characterized. Here, we conducted a characterization of organelle-type AtNUDX proteins (AtNUDX12 to -24, -26, and -27). AtNUDX14 showed pyrophosphohydrolase activity toward both ADP-ribose and ADP-glucose, although its K  m value was approximately 100-fold lower for ADP-ribose (13.0 ± 0.7 μ  m) than for ADP-glucose (1,235 ± 65 μ  m). AtNUDX15 hydrolyzed not only reduced coenzyme A (118.7 ± 3.4 μ  m) but also a wide range of its derivatives. AtNUDX19 showed pyrophosphohydrolase activity toward both NADH (335.3 ± 5.4 μ  m) and NADPH (36.9 ± 3.5 μ  m). AtNUDX23 had flavin adenine dinucleotide pyrophosphohydrolase activity (9.1 ± 0.9 μ  m). Both AtNUDX26 and AtNUDX27 hydrolyzed diadenosine polyphosphates (n = 4–5). A confocal microscopic analysis using a green fluorescent protein fusion protein showed that AtNUDX15 is distributed in mitochondria and AtNUDX14 -19, -23, -26, and -27 are distributed in chloroplasts. These AtNUDX mRNAs were detected ubiquitously in various Arabidopsis tissues. The T-DNA insertion mutants of AtNUDX13, -14, -15, -19, -20, -21, -25, -26, and -27 did not exhibit any phenotypical differences under normal growth conditions. These results suggest that Nudix hydrolases in Arabidopsis control a variety of metabolites and are pertinent to a wide range of physiological processes.
Crystal structures of Mycobacterium tuberculosis S‐adenosyl‐L‐homocysteine hydrolase in ternary complex with substrate and inhibitors
Authors: Reddy Manchi C.M. , Kuppan Gokulan , Shetty Nishant D. , Owen Joshua L. , Ioerger Thomas R. , Sacchettini James C.
Published: September 2008

S‐adenosylhomocysteine hydrolase (SAHH) is a ubiquitous enzyme that plays a central role in methylation‐based processes by maintaining the intracellular balance between S‐adenosylhomocysteine (SAH) and S‐adenosylmethionine. We report the first prokaryotic crystal structure of SAHH, from Mycobacterium tuberculosis (Mtb), in complex with adenosine (ADO) and nicotinamide adenine dinucleotide. Structures of complexes with three inhibitors are also reported: 3′‐keto aristeromycin (ARI), 2‐fluoroadenosine, and 3‐deazaadenosine. The ARI complex is the first reported structure of SAHH complexed with this inhibitor, and confirms the oxidation of the 3′ hydroxyl to a planar keto group, consistent with its prediction as a mechanism‐based inhibitor. We demonstrate the in vivo enzyme inhibition activity of the three inhibitors and also show that 2‐fluoradenosine has bactericidal activity. While most of the residues lining the ADO‐binding pocket are identical between Mtb and human SAHH, less is known about the binding mode of the homocysteine (HCY) appendage of the full substrate. We report the 2.0 Å resolution structure of the complex of SAHH cocrystallized with SAH. The most striking change in the structure is that binding of HCY forces a rotation of His363 around the backbone to flip out of contact with the 5′ hydroxyl of the ADO and opens access to a nearby channel that leads to the surface. This complex suggests that His363 acts as a switch that opens up to permit binding of substrate, then closes down after release of the cleaved HCY. Differences in the entrance to this access channel between human and Mtb SAHH are identified.
Effects of Lactobacillus GG treatment during pregnancy on the development of fetal antigen‐specific immune responses
Authors: Boyle R. J. , Mah L‐J. , Chen A. , Kivivuori S. , Robins‐Browne R. M. , Tang M. L‐K.
Published: September 2008

Background
Several clinical trials suggest that probiotics may have a role in the prevention of eczema. The optimal timing and mechanisms underlying this intervention are not clear. In particular it is not known whether such treatment works during pregnancy or whether postnatal exposure is important.

Objective
We investigated whether the probiotic Lactobacillus rhamnosus strain GG (LGG) influences fetal immune responses when administered to pregnant women, as a possible mechanism for its protective effects against the development of eczema.

Methods
Peripheral blood mononuclear cell from 11 adults treated with LGG, and cord blood mononuclear cells (CBMCs) from 73 women participating in a randomized controlled trial of LGG treatment were cultured with heat‐killed LGG, ovalbumin (OVA) or without stimulus. Cells were analysed by flow cytometry and real‐time PCR for markers of dendritic cell (DC) phenotype, T cell proliferation and regulation. Cytokine secretion was analysed in culture supernatants by multiplex cytokine assay.

Results
LGG treatment of adults led to systemic immune responses suggestive of antigen‐specific tolerance including reduced CD4+ T cell proliferation to heat‐killed LGG (30% reduction; P=0.03). LGG treatment of pregnant women did not influence CD4+ T cell proliferation, forkhead box P3 expression, DC phenotype or cytokine secretion in CBMCs cultured with heat‐killed LGG or OVA.

Conclusion
LGG treatment of pregnant women fails to influence fetal antigen‐specific immune responses. This suggests that modulation of fetal immune responses may not be a major mechanism by which probiotics such as LGG prevent eczema.
Intravenous zoledronate improves bone density in adults with cystic fibrosis (CF)
Authors: Chapman Ian , Greville Hugh , Ebeling Peter R. , King Susannah J. , Kotsimbos Tom , Nugent Patricia , Player Rosie , Topliss Duncan J. , Warner Janet , Wilson John W.
Published: September 2008

Objective  Reduced bone mineral density (BMD) and increased rates of atraumatic fracture are observed in cystic fibrosis (CF) patients, causing increasing morbidity as this population ages. The study aimed to assess the safety, tolerability and effect on BMD of intravenous zoledronate in adults with CF and osteopaenia.

Design  Randomized, double‐blind, placebo‐controlled clinical trial.

Setting  Adult CF outpatient clinics at two hospitals.

Patients  Twenty‐two non‐transplanted CF patients aged ≥ 18 years with a bone densitometry T‐score of < –1·5 at one of three sites (lumbar spine, femoral neck, distal forearm) were studied. Participants were randomized to receive either 2 mg zoledronate IV (n = 10) or normal saline (placebo, n = 12) every 3 months for 2 years (8 infusions). All participants received calcium and vitamin D supplements twice daily.

Measurements  Percentage change in areal BMD from baseline.

Results  Lumbar spine BMD increased from baseline more with zoledronate than placebo at 6 months (5·35 ± 0·76 vs. 1·19 ± 1·20%, P = 0·012), 12 months (6·6 ± 1·5 vs. 0·35 ± 1·55%, P = 0·011) and 24 months (6·14 ± 1·86 vs. 0·44 ± 0·10, P = 0·021). Femoral neck BMD increased more after zoledronate than placebo at 6 months (3·2 ± 1·6 vs.–1·43 ± 0·43%, P = 0·019), 12 months (4·12 ± 1·8 vs.–1·59 ± 1·4%, P = 0·024) and 24 months (4·23 ± 1·3 vs.–2·5 ± 1·41%, P = 0·0028). Forearm BMD did not change. Zoledronate was associated with flu‐like and musculoskeletal side effects, particularly after the first infusion. There were no fractures in either group.

Conclusion  Intravenous zoledronate was significantly more effective than placebo for increasing BMD in adults with CF and osteopaenia, but side effects limited its tolerability.
Cross‐sectional analysis of testosterone therapies in hypopituitary men on stable pituitary hormone replacement
Authors: Siyambalapitiya S. , Jonsson P. , Koltowska‐Haggstrom M. , Gaillard R. , Ho K. , Ross R. J. M.
Published: September 2008

Objective  The last decade has seen a proliferation in options for testosterone replacement. However, little is known as to the benefits of different treatment modalities. Our objective was to determine the testosterone prescription pattern and to examine the impact on various outcome measures.

Subjects and methods  A total of 816 adult‐onset hypopituitary males on stable pituitary replacement for at least 1 year were identified from the KIMS database. Patients were classified as either eugonadal (n = 106), or hypogonadal (n = 710) on intramuscular (IM, n = 558), oral (n = 74), transdermal (n = 61), and depot (n = 17) testosterone.

Results  After 1 year of stable pituitary replacement therapy, body composition, cardiovascular parameters, GH replacement and quality of life were not significantly different in androgen‐replaced hypogonadal patients compared to eugonadal patients. There were no differences in outcome variables within the hypogonadal group according to the testosterone replacement regimen used and no difference in response to GH therapy.

Conclusions  The majority of hypopituitary patients in the last decade have received IM testosterone. Body composition, cardiovascular parameters, GH replacement and quality of life were not different between eugonadal and hypogonadal patients and were not differentially affected by the mode of testosterone replacement. These findings are reassuring that there is no major difference in response to different testosterone replacement regimens.
Selective COX2 inhibition improves whole body and muscular insulin resistance in fructose‐fed rats
Authors: Hsieh P.‐S. , Tsai H.‐C. , Kuo C.‐H. , Chan J. Y.‐H. , Shyu J.‐F. , Cheng W.‐T. , Liu T.‐T.
Published: September 2008

Background  The effects of cyclooxygenase‐1 (COX1) and cyclooxygenase‐2 (COX2) inhibition on insulin resistance in subjects with the metabolic syndrome remain elusive. Aims of this study were to examine the effects of COX1 and COX2 inhibitors on whole body and muscular insulin resistance in fructose‐fed rats, an animal model of the metabolic syndrome.

Materials and methods  The rats on regular or 60% fructose‐enriched diets for 6 weeks were further divided into rats combined with or without piroxicam (a selective COX1 inhibitor) or celecoxib (a selective COX2 inhibitor) treatment for an additional 2 weeks. Euglycaemic hyperinsulinaemic clamp (EHC) with a tracer dilution method was performed at the end of the study.

Results  The present result showed that fructose‐induced increases in systolic blood pressure and fasting plasma insulin levels were significantly suppressed in rats treated with celecoxib but not piroxicam. In the EHC period, celecoxib significantly reversed fructose‐induced decreases in whole body glucose uptake, mainly by glucose storage. Hepatic glucose production and whole body glycolysis were not significantly changed among groups. Celecoxib but not piroxicam significantly reversed fructose‐induced decreases in glycogen synthase activities in red and white quadriceps muscles and insulin‐stimulated membrane GLUT4 recruitment in soleus muscles. Celecoxib and piroxicam both significantly diminished fructose‐induced increases in plasma thromboxane B2 and 6‐keto prostaglandin (PG) F1α; but only celecoxib treatment significantly attenuated a fructose‐induced increase in 8‐isoprostane levels. Plasma PGE metabolites were not different among groups.

Conclusions  This study demonstrates that a therapeutic dose of celecoxib, but not piroxicam, could significantly attenuate fructose‐induced whole body and muscular insulin resistance in rats.
Remote estimation of carbon dioxide uptake by a Mediterranean forest
Authors: GARBULSKY MARTÍN F. , PEÑUELAS JOSEP , PAPALE DARIO , FILELLA IOLANDA
Published: September 2008

The estimation of the carbon balance in ecosystems, regions, and the biosphere is currently one of the main concerns in the study of the ecology of global change. Current remote sensing methodologies for estimating gross primary productivity are not satisfactory because they rely too heavily on (i) the availability of climatic data, (ii) the definition of land‐use cover, and (iii) the assumptions of the effects of these two factors on the radiation‐use efficiency of vegetation (RUE). A new methodology is urgently needed that will actually assess RUE and overcome the problems associated with the capture of fluctuations in carbon absorption in space and over time. Remote sensing techniques such as the widely used reflectance vegetation indices (e.g. NDVI, EVI) allow green plant biomass and therefore plant photosynthetic capacity to be assessed. However, there are vegetation types, such as the Mediterranean forests, with a very low seasonality of these vegetation indices and a high seasonality of carbon uptake. In these cases it is important to detect how much of this capacity is actually realized, which is a much more challenging goal. The photochemical reflectance index (PRI) derived from freely available satellite information (MODIS sensor) presented for a 5‐year analysis for a Mediterranean forest a positive relationship with the RUE. Thus, we show that it is possible to estimate RUE and GPP in real time and therefore actual carbon uptake of Mediterranean forests at ecosystem level using the PRI. This conceptual and technological advancement would avoid the need to rely on the sometimes unreliable maximum RUE.
Effects of soil frost on soil respiration and its radiocarbon signature in a Norway spruce forest soil
Authors: MUHR JAN , BORKEN WERNER , MATZNER EGBERT
Published: September 2008

Apart from a general increase of mean annual air temperature, climate models predict a regional increase of the frequency and intensity of soil frost with possibly strong effects on C cycling of soils. In this study, we induced mild soil frost (up to −5 °C in a depth of 5 cm below surface) in a Norway spruce forest soil by removing the natural snow cover in the winter of 2005/2006. Soil frost lasted from January to April 2006 and was detected down to 15 cm depth. Soil frost effectively reduced soil respiration in the snow removal plots in comparison to undisturbed control plots. On an annual basis 6.2 t C ha−1 a−1 were emitted in the control plots compared with 5.1 t C ha−1 a−1 in the snow removal plots. Only 14% of this difference was attributed to reduced soil respiration during the soil frost period itself, whereas 63% of this difference originated from differences during the summer of 2006. Radiocarbon (Δ14C) signature of CO2 revealed a considerable reduction of heterotrophic respiration on the snow removal plots, only partly compensated for by a slight increase of rhizosphere respiration. Similar CO2 concentrations in the uppermost mineral horizons of both treatments indicate that differences between the treatments originated from the organic horizons. Extremely low water contents between June and October of 2006 may have inhibited the recovery of the heterotrophic organisms from the frost period, thereby enhancing the differences between the control and snow removal plots. We conclude that soil frost triggered a change in the composition of the microbial community, leading to an increased sensitivity of heterotrophic respiration to summer drought. A CO2 pulse during thawing, such as described for arable soils several times throughout the literature, with the potential to partly compensate for reduced soil respiration during soil frost, appears to be lacking for this soil. Our results from this experiment indicate that soil frost reduces C emission from forest soils, whereas mild winters may enhance C losses from forest soils.
Experiences in the prevention of arthropathy in haemophila patients with inhibitors
Authors: JIMENEZ‐YUSTE V. , RODRIGUEZ‐MERCHAN E. C. , ALVAREZ M. T. , QUINTANA M. , MARTIN‐SALCES M. , HERNANDEZ‐NAVARRO F.
Published: September 2008

Summary. Haemophilia patients with inhibitor have a higher level of arthropathy and more severe joint morbidity than patients without inhibitors. In recent years, interest has grown in the possibility that bypassing agent regimens could prevent bleeding and, consequently, arthropathy in inhibitor patients. Nevertheless, doubts about efficacy, complications and cost exist, questioning the justification of an uncertain prophylaxis in patients with inhibitors. Activated prothrombin complex concentrate (aPCC) has been used in more than 70 haemophilia patients with inhibitors in different clinical situations. aPCC prophylaxis seems to be safe and effective for the reduction of bleeding episodes in some patients. Recombinant activated factor VII (rFVIIa) has been employed prophylactically in over 44 haemophilia patients with inhibitors; 22 patients were included in the only randomized, prospective clinical trial of bypassing agents in prophylaxis. Bleeding frequency was reduced and this reduction was maintained during the postprophylaxis period. No thromboembolic events were reported during prophylaxis with rFVIIa. Although the effect of aPCC can last longer than that of rFVIIa, their efficacy rates are similar, suggesting that the biological effect of rFVIIa is actually much longer than indicated by its short plasma half‐life. aPCC contains residual factor VIII antigen and may cause an anamnestic response in the inhibitor titre. This is crucial when immune tolerance induction is postponed to allow the inhibitor titre to decline to <10 Bethesda Units. In this setting, aPCC is not recommended as a first‐line prophylaxis because of its potential to protract anamnesis, and rFVIIa is the preferred agent.
Telephone helpline for parents of children with congenital anomalies
Authors: Gischler Saskia J. , Mazer Petra , Poley Marten J. , Tibboel Dick , Van Dijk Monique
Published: September 2008

Title.  Telephone helpline for parents of children with congenital anomalies.

Aim.  This paper is a report of a study to evaluate how often and for what reason parents of children born with severe anatomical congenital anomalies use a 24‐hour telephone helpline, and to identify differences between callers and non‐callers.

Background.  Children born with severe congenital anomalies often remain dependent on medical care and technology after discharge. Adequate medical consultation in the home situation may lower parental burden.

Method.  Observational study of telephone contacts from 2000 to 2006 with parents of congenital anomaly patients discharged home after neonatal intensive care admission. Frequency of telephone calls was categorized per type of anomaly. Resulting interventions in terms of consultation and hospital admission were recorded. Finally, costs of personnel needed to provide 24‐hour telephone helpline availability were calculated.

Findings.  A total of 670 calls occurred outside office hours, from 34·4% of all parents; 23·7% of these calls concerned feeding problems. Parents of children with oesophageal atresia, urogenital malformation and congenital diaphragmatic hernia called most frequently (44·3–50·6%). Non‐callers were more often first‐time parents, divorced/separated or immigrants. Nurses handled 24·5% of calls by themselves and 20·2% of calls led to emergency room consultations resulting in 4·9% admissions. First contact took place at median age 3 months, last contact at median age 8 months. Total personnel costs amounted to €27,191 per year.

Conclusion.  A 24‐hour telephone helpline provides easy access to medical and supportive care for parents of children with congenital anomalies at relatively low cost. Nurses can effectively run this telephone helpline with paediatrician back‐up.
Quantifying cell–matrix adhesion dynamics in living cells using interference reflection microscopy
Authors: HOLT M.R. , CALLE Y. , SUTTON D.H. , CRITCHLEY D.R. , JONES G.E. , DUNN G.A.
Published: September 2008

Focal adhesions and podosomes are integrin‐mediated cell‐substratum contacts that can be visualized using interference reflection microscopy (IRM). Here, we have developed automated image‐processing procedures to quantify adhesion turnover from IRM images of live cells. Using time sequences of images, we produce adhesion maps that reveal the spatial changes of adhesions and contain additional information on the time sequence of these changes. Such maps were used to characterize focal adhesion dynamics in mouse embryo fibroblasts lacking one or both alleles of the vinculin gene. Loss of vinculin expression resulted in increased assembly, disassembly and/or in increased translocation of focal adhesions, suggesting that vinculin is important for stabilizing focal adhesions. This method is also useful for studying the rapid dynamics of podosomes as observed in primary mouse dendritic cells.
Mechanical properties of plasma membrane and nuclear envelope measured by scanning probe microscope
Authors: YOKOKAWA M. , TAKEYASU K. , YOSHIMURA S.H.
Published: September 2008

Atomic force microscopy has been used to visualize nano‐scale structures of various cellular components and to characterize mechanical properties of biomolecules. In spite of its ability to measure non‐fixed samples in liquid, the application of AFM for living cell manipulation has been hampered by the lack of knowledge of the mechanical properties of living cells. In this study, we successfully combine AFM imaging and force measurement to characterize the mechanical properties of the plasma membrane and the nuclear envelope of living HeLa cells in a culture medium. We examine cantilevers with different physical properties (spring constant, tip angle and length) to find out the one suitable for living cell imaging and manipulation. Our results of elasticity measurement revealed that both the plasma membrane and the nuclear envelope are soft enough to absorb a large deformation by the AFM probe. The penetrations of the plasma membrane and the nuclear envelope were possible when the probe indents the cell membranes far down close to a hard glass surface. These results provide useful information to the development of single‐cell manipulation techniques.
Image matching between experimental and simulated high‐resolution electron micrographs of sapphire on the orientation
Authors: DU K. , RÜHLE M.
Published: September 2008

The effects of imaging parameters have been studied on their roles of the severe mismatches between experimental and simulated high‐resolution transmission electron micrographs of sapphire along the direction. Image simulation and convergent‐beam electron diffraction techniques have been performed on misalignments of the electron beam and the crystal specimen. Based on this study, we have introduced an approach to achieve reliable simulation for experimental images of sapphire on the projection by the use of iterative digital image matching.
Sub‐100‐nanometre resolution in total internal reflection fluorescence microscopy
Authors: BECK M. , ASCHWANDEN M. , STEMMER A.
Published: September 2008

Combining total internal reflection fluorescence microscopy with structured illumination allows optical wide‐field imaging with sub‐100‐nanometre resolution. We present a novel objective‐launch set‐up for standing wave illumination that takes advantage of a tunable transmission diffraction grating and transparent phase shifters actuated by electro‐active polymers to control the excitation pattern in three dimensions. Image acquisition is completed in less than 1 s. To reconstruct the extended image spectrum, we apply a new apodization function that results in a lateral resolution of 89 nm for green emission wavelength.
Alignment with sub‐pixel accuracy for images of multi‐modality microscopes using automatic calibration
Authors: TSAI C.‐L. , WARGER II W.C. , LAEVSKY G.S. , DIMARZIO C.A.
Published: September 2008

A biological specimen is often imaged with various imaging modalities, and it is crucial that such images are well aligned to best reveal physiological structures and functions of the specimen for in‐depth analyses. In this paper, we present a methodology for automatic calibration of multiple optical imaging modalities within the x–y detector plane using a custom chrome‐on‐glass target and an automatic and accurate registration algorithm. The target contains lines crossing at random angles, and our method of registration is based on the alignment of salient features extracted from the lines within the individual images. Once spatial relationships are found between the various detectors and applied to the resultant images, no further registration is required for all static samples, and the registered images serve as the starting point for registration of dynamic samples, where the remaining misalignment is caused by sample movement. We have validated our algorithm with 40 inter‐modal and 30 intra‐modal image pairs, and the success rates are 95 and 100%, respectively, with sub‐pixel accuracy. This methodology is widely applicable to any multi‐modal microscope that combines a number of imaging modalities on a common platform assuming images of the target can be obtained.
Quantitative tomography of early mouse embryos: laser scanning microscopy and 3D reconstruction
Authors: POGORELOV A.G. , POGORELOVA V.N.
Published: September 2008

The cell volume alteration participates in a wide variety of cellular functions that may interfere with intra‐cellular homeostasis. The most adequate approach of estimation of the volume changes induced by osmotic misbalance, alteration in shape and size due to the action shape forming substances, etc., is the direct measurement of volumetric parameters of embryos. In the given research, the volume magnitude and kinetics of changes in volume and surface area of blastomere and polar bodies of early mouse embryos were determined using three‐dimensional reconstruction of the optical section stack obtained with laser scanning microscope (LSM). The size and surface area were determined for isotonic and anisosmotic conditions. The physiological significance of the findings is discussed.
Advantages and pitfalls of using free‐hand sections of frozen needles for three‐dimensional analysis of mesophyll by stereology and confocal microscopy
Authors: LHOTÁKOVÁ Z. , ALBRECHTOVÁ J. , JANÁČEK J. , KUBÍNOVÁ L.
Published: September 2008

The anatomical structure of mesophyll tissue in the leaf is tightly connected with many physiological processes in plants. One of the most important mesophyll parameters related to photosynthesis is the internal leaf surface area, i.e. the surface area of mesophyll cell walls exposed to intercellular spaces. An efficient design‐based stereological method can be applied for estimation of this parameter, using software‐randomized virtual fakir test probes in stacks of optical sections acquired by a confocal microscope within thick physical free‐hand sections (i.e. acquired using a hand microtome), as we have shown in the case of fresh Norway spruce needles recently. However, for wider practical use in plant ecophysiology, a suitable form of sample storage and other possible technical constraints of this methodology need to be checked. We tested the effect of freezing conifer needles on their anatomical structure as well as the effect of possible deformations due to the cutting of unembedded material by a hand microtome, which can result in distortions of cutting surfaces. In the present study we found a higher proportion of intercellular spaces in mesophyll in regions near to the surface of a physical section, which means that the measurements should be restricted only to the middle region of the optical section series. On the other hand, the proportion of intercellular spaces in mesophyll as well as the internal needle surface density in mesophyll did not show significant difference between fresh and frozen needles; therefore, we conclude that freezing represents a suitable form of storage of sampled material for proposed stereological evaluation.
Stereological estimation for layered structures based on slabs perpendicular to a surface
Authors: LEGLAND D. , DEVAUX M.‐F. , KIÊU K. , BOUCHET B.
Published: September 2008

This paper deals with the characterization of layered structures sampled with respect to a reference surface. A scheme where thick slabs are sampled perpendicular to a curved surface is considered, resulting in a non‐uniform sampling of the structure. We present an estimation procedure based on the Horvitz–Thompson principle. An approximation of the sampling probability is proposed, which depends on the local surface curvatures, on the slab dimensions and on the intensity function of slab anchors. The practical determination of local parameters is detailed for the case of a revolution surface. The procedure is applied to the estimation of surface area density of cell walls in tomato pericarp.
Subcellular imaging of RNA distribution and DNA replication in single mammalian cells with SIMS: the localization of heat shock induced RNA in relation to the distribution of intranuclear bound calcium
Authors: CHANDRA S.
Published: September 2008

The subcellular localization of RNA for understanding transcriptional activity by using RNA precursors, like 5‐bromouridine (BrU), generally requires chemical fixation and staining of cells with monoclonal antibody for imaging BrU‐containing RNA in individual cells. Although effective for RNA localization, the native chemical composition of diffusible ions and molecules is destroyed in this approach and one cannot study their spatial relationship with RNA localization sites in this sample type. This work presents a novel secondary ion mass spectrometry approach in cryogenically prepared cells, which allows the same cell imaging of RNA (and/or replicating DNA) distribution in relation to intracellular chemical composition. The heat shock treatment of HeLa cells was used as a model system because the transcription of heat shock genes is activated during heat shock while other transcriptional activities of the cell are suppressed. The HeLa cells were heat‐shocked for 1 h at 42°C in presence of 100 μM BrU and/or 100 μM IdU (5‐iododeoxyuridine). Following the heat shock treatments, the cells were cryogenically prepared with our sandwich freeze‐fracture method and freeze‐dried prior to secondary ion mass spectrometry analysis. A CAMECA IMS 3f secondary ion mass spectrometry ion microscope (CAMECA, Paris, France) capable of producing elemental (isotopic) distributions with a spatial resolution of 500 nm was used in the study. Secondary ion mass spectrometry analysis of fractured freeze‐dried HeLa cells revealed well‐preserved intracellular 39K and 23Na concentrations in heat‐shocked cells. Both DNA replication and RNA distribution (total RNA) were imaged directly in the same cell by secondary ion mass spectrometry imaging of masses 127I (from IdU) and 81Br (from BrU), respectively. Surprisingly, the nucleus of heat‐shocked cells contained spatially resolved regions with elevated levels of bound calcium (approximately 0.75 mM total calcium instead of 0.50 mM total calcium in the nucleoplasm). These regions spatially correlated with depleted levels of BrU‐RNA in 81Br secondary ion mass spectrometry images. The remainder of intranuclear regions displayed the presence of BrU‐RNA with heterogeneous distribution. These observations indicate that calcium in its bound form may play a fundamental role in processes such as transcription and/or processing and storage of RNA. The shape of intranuclear regions with elevated levels of bound calcium resembled the heat shock induced nuclear bodies in HeLa cells. The analysis of cryogenically prepared frozen freeze‐dried cells provides an ideal sample type for further understanding of the role of bound calcium in transcription of genes under physiological and pathological conditions.
Comparison of three cell fixation methods for high content analysis assays utilizing quantum dots
Authors: WILLIAMS Y. , BYRNE S. , BASHIR M. , DAVIES A. , WHELAN Á. , GUN’KO Y. , KELLEHER D. , VOLKOV Y.
Published: September 2008

Semiconductor nanoparticles or quantum dots are being increasingly utilized as fluorescent probes in cell biology both in live and fixed cell assays. Quantum dots possess an immense potential for use in multiplexing assays that can be run on high content screening analysers. Depending on the nature of the biological target under investigation, experiments are frequently required on cells retaining an intact cell membrane or also on those that have been fixed and permeabilized to expose intracellular antigens. Fixation of cell lines before or after the addition of quantum dots may affect their localization, emission properties and stability. Using a high content analysis platform we perform a quantitative comparative analysis of three common fixation techniques in two different cell lines exposed to carboxylic acid stabilized CdTe quantum dots.

Our study demonstrates that in prefixed and permeabilized cells, quantum dots are readily internalized regardless of cell type, and their intracellular location is primarily determined by the properties of the quantum dots themselves. However, if the fixation procedures are preformed on live cells previously incubated with quantum dots, other important factors have to be considered. The choice of the fixative significantly influences the fluorescent characteristics of the quantum dots. Fixatives, regardless of their chemical nature, negatively affected quantum dots fluorescence intensity. Comparative analysis of gluteraldehyde, methanol and paraformaldehyde demonstrated that 2% paraformaldehyde was the fixative of choice. The presence of protein in the media did not significantly alter the quantum dot fluorescence.

This study indicates that multiplexing assays utilizing quantum dots, despite being a cutting edge tool for high content cell imaging, still require careful consideration of the basic steps in biological sample processing.
TEM analysis of centreline sulphide precipitates modified by titanium additions to low carbon steel
Authors: AMINORROAYA S. , DIPPENAAR R.
Published: September 2008

Elongated inclusions, particularly MnS, contribute significantly to reduced ductility and toughness in hot rolled steel but earlier research indicated that these properties can be improved by titanium additions. Such additions to a steel result in titanium being dissolved in manganese sulphide or MnS being replaced by TiS and/or titanium carbosulphides. In the present study, a steel was designed to decrease alloying element segregation and to evaluate the effect of titanium on centreline sulphide precipitates. Precipitates were identified by using scanning electron microscopy and characterized by the use of transmission electron microscopy following sample preparation by focused ion beam milling techniques. Iron–titanium‐sulphides form in close proximity to MnS precipitates that contain iron. Evidence is provided that an increase in the titanium content of steel leads to an increase in the percentage of titanium contained in the iron sulphides and a decrease in the iron content of MnS inclusions.