bluuebunny/crossref_metadata_2025 huggingface dataset:
Population-Based Surveillance of Clostridium difficile Infection in Manitoba, Canada, by Using Interim Surveillance Definitions
Authors: Lambert Pascal J. , Dyck Myrna , Thompson Laura H. , Hammond Greg W.
Published: September 2009

Objective.

TO apply interim surveillance definitions of Clostridium difficile infection (CDI) cases to 1 year of data from the provincewide surveillance system of Manitoba, Canada, to determine the epidemiology of CDI incident cases in a population.

Methods.

CDI cases were categorized with interim surveillance definitions developed by an ad hoc C. difficile surveillance working group. Incident cases recorded in the provincial CDI database between July 2005 and June 2006 were linked to the provincial hospitalization and nursing home databases and analyzed.

Results.

One thousand six incident cases were identified over 1 year. Five hundred fifteen (51%) cases were associated with and began in a healthcare facility (HCF), whereas 275 (27%) were associated with and began in the community. An additional 131 (13%) cases were HCF associated but began in the community, while 85 (8%) were of indeterminate origin. Cases of HCF-associated CDI occurred in patients who were older than did cases of community-associated CDI (P < .0001). The provincial rate of community-onset cases was 23.4 per 100,000 person-years, and rates varied among geographic areas. HCF-associated CDI rates among the 10 largest hospitals varied from 0.5 to 8.4 per 10,000 patient-days. The time to CDI onset after hospital admission indicated that 25% of nosocomial cases began by the 8th day, and 50% began by the 17th day.

Conclusions.

Although the majority of CDI cases were associated with exposure to a HCF, 40% of incident CDI began in the community. Populations with HCF- and community-associated CDI demonstrated significantly different age distributions. The wide variation of rates among HCFs requires explanation. The high percentage of incident cases in the community warrants increased study.
STELLAR AND TOTAL BARYON MASS FRACTIONS IN GROUPS AND CLUSTERS SINCE REDSHIFT 1*
Authors: Giodini S. , Pierini D. , Finoguenov A. , Pratt G. W. , Boehringer H. , Leauthaud A. , Guzzo L. , Aussel H. , Bolzonella M. , Capak P. , Elvis M. , Hasinger G. , Ilbert O. , Kartaltepe J. S. , Koekemoer A. M. , Lilly S. J. , Massey R. , McCracken H. J. , Rhodes J. , Salvato M. , Sanders D. B. , Scoville N. Z. , Sasaki S. , Smolcic V. , Taniguchi Y. , Thompson D.
Published: September 2009

We investigate if the discrepancy between estimates of the total baryon mass fraction obtained from observations of the cosmic microwave background (CMB) and of galaxy groups/clusters persists when a large sample of groups is considered. To this purpose, 91 candidate X-ray groups/poor clusters at redshift 0.1 ⩽ z ⩽ 1 are selected from the COSMOS 2 deg2 survey, based only on their X-ray luminosity and extent. This sample is complemented by 27 nearby clusters with a robust, analogous determination of the total and stellar mass inside R
500. The total sample of 118 groups and clusters with z ⩽ 1 spans a range in M
500 of ∼1013–1015
M
☉. We find that the stellar mass fraction associated with galaxies at R
500 decreases with increasing total mass as M
−0.37 ± 0.04
500, independent of redshift. Estimating the total gas mass fraction from a recently derived, high-quality scaling relation, the total baryon mass fraction (f
stars+gas
500 = f
stars
500 + f
gas
500) is found to increase by ∼25%, when M
500 increases from 〈M〉 = 5 × 1013
M
☉ to 〈M〉 = 7 × 1014
M
☉. After consideration of a plausible contribution due to intracluster light (11%–22% of the total stellar mass) and gas depletion through the hierarchical assembly process (10% of the gas mass), the estimated values of the total baryon mass fraction are still lower than the latest CMB measure of the same quantity (WMAP5), at a significance level of 3.3σ for groups of 〈M〉 = 5 × 1013
M
☉. The discrepancy decreases toward higher total masses, such that it is 1σ at 〈M〉 = 7 × 1014
M
☉. We discuss this result in terms of nongravitational processes such as feedback and filamentary heating.
Influence of Craniosacral Therapy on Anxiety, Depression and Quality of Life in Patients with Fibromyalgia
Authors: Matarán-Peñarrocha Guillermo A. , Castro-Sánchez Adelaida María , García Gloria Carballo , Moreno-Lorenzo Carmen , Carreño Tesifón Parrón , Zafra María Dolores Onieva
Published: September 2009

Fibromyalgia is considered as a combination of physical, psychological and social disabilities. The causes of pathologic mechanism underlying fibromyalgia are unknown, but fibromyalgia may lead to reduced quality of life. The objective of this study was to analyze the repercussions of craniosacral therapy on depression, anxiety and quality of life in fibromyalgia patients with painful symptoms. An experimental, double‐blind longitudinal clinical trial design was undertaken. Eighty‐four patients diagnosed with fibromyalgia were randomly assigned to an intervention group (craniosacral therapy) or placebo group (simulated treatment with disconnected ultrasound). The treatment period was 25 weeks. Anxiety, pain, sleep quality, depression and quality of life were determined at baseline and at 10 minutes, 6 months and 1‐year post‐treatment. State anxiety and trait anxiety, pain, quality of life and Pittsburgh sleep quality index were significantly higher in the intervention versus placebo group after the treatment period and at the 6‐month follow‐up. However, at the 1‐year follow‐up, the groups only differed in the Pittsburgh sleep quality index. Approaching fibromyalgia by means of craniosacral therapy contributes to improving anxiety and quality of life levels in these patients.
Characterization of Resistant Starch Samples Prepared from Two High‐Amylose Maize Starches Through Debranching and Heat Treatments
Authors: Ozturk Serpil , Koksel Hamit , Ng Perry K. W.
Published: September 2009

The aim of the present study was to investigate effects of debranching, autoclaving‐storing cycles, and drying processes (oven‐drying or freeze‐drying) on RS contents, thermal, pasting, and functional properties of high‐amylose maize starches (Hylon V and Hylon VII). The resistant starch (RS) contents increased (≤57.8%) with increasing autoclaving‐storing cycles. RS contents of oven‐dried samples were higher than those of freeze‐dried samples due to ongoing retrogradation of starch during oven drying at 50°C. Debranching caused a significant decrease in peak transition temperature and enthalpy values as compared with native starches. Solubility and water binding values of RS preparations were higher than those of native starches. Addition of native and autoclaved samples had improving effect on emulsion properties of albumin. Cold viscosity values of oven‐dried samples were lower as compared with freeze‐dried samples; this might be due to higher number of H‐bonds in the oven‐dried samples expected to be formed during drying. Debranching and autoclaving‐storing cycles caused decreases in peak, breakdown, and final viscosity values. The results of present study showed that debranching and heat treatments increased the RS contents and improved the functional properties of high‐amylose maize starches.
Effect of Temperature and Steeping Time on Calcium and Phosphorus Content in Nixtamalized Corn Flours Obtained by Traditional Nixtamalization Process
Authors: Rojas‐Molina I. , Gutiérrez E. , Rojas A. , Cortés‐Álvarez M. , Campos‐Solís L. , Hernández‐Urbiola M. , Arjona J. L. , Cornejo A. , Rodríguez‐García M. E.
Published: September 2009

This report shows the effect of temperature (72, 82, and 92°C) during the cooking stage and steeping time (0, 1, 3, 5, 7, 9, 11, 13, and 15 hr) on calcium and phosphorus contents in nixtamalized corn flours obtained by the traditional nixtamalization process (NCF). In addition, calcium and phosphorus contents in industrial nixtamalized corn flours were analyzed for comparative purposes. Atomic absorption spectroscopy and UV‐vis spectroscopy methods were used to study the calcium and phosphorus contents as well as the Ca2+/P ratio in NCF and industrial nixtamalized corn flours. Additionally, deposition and identification of calcium compounds in the nixtamalized corn pericarp were analyzed by low‐vacuum scanning electron microscopy, energy dispersive spectrometry, and X‐ray diffraction techniques. Dry matter loss in NCF is also reported. As the temperature increased, Ca2+ content was enhanced, while the phosphorus content decreased with statistical differences (P ≤ 0.05) between thermal treatments. Ca2+ content in industrial nixtamalized corn flours was significantly lower (P ≤ 0.05) than that of NCF. On the other hand, no statistical differences (P ≤ 0.05) were found between phosphorus content in commercial nixtamalized corn flours and NCF. Calcium compounds, identified as calcite, were detected in corn pericarp. Statistical differences (P ≤ 0.05) were observed in phosphorous content in NCF obtained at different cooking temperatures. In addition, a decrease in phosphorus levels significantly correlated with the steeping time at 92°C (r = –0.91). At 72, 82, and 92°C, the average Ca2+/P ratio in NCF was 0.45 ± 0.03, 0.61 ± 0.05, and 0.82 ± 0.05, respectively, indicating a correlation between this parameter and the cooking temperature. However, no correlation was found between the Ca2+/P ratio and the steeping time. This behavior is attributed to calcium attached to corn kernel. In commercial nixtamalized corn flours, the Ca2+/P ratio was significantly lower (P ≤ 0.05) than that of NCF. There was a significant correlation (P ≤ 0.01) between dry matter loss and steeping time (r = 0.99) in NCF, this fact influenced the Ca2+/P ratio due to the calcium attached to pericarp. At 82 and 92°C, maximum values of Ca2+/P ratio were detected in NCF at 7 hr of steeping time and at 9 hr at 72°C. These results can be used with industrial purposes to assess a maximum calcium‐to‐phosphorus ratio, and at the same time, to avoid the loss of pericarp to increase the functional properties of NCF.
The ins and outs of extracellular matrix assembly
Authors: Kadler Karl E.
Published: September 2009

Introduction  To a rough approximation, the adult human comprises 2–5 kg of cells. The remaining mass originates from the extracellular matrix (ECM). The ECM is highly organized, which is surprising considering the paucity of cells and the fact that the ECM comprises some of the largest and most insoluble macromolecules encoded by the genome. The high level of supramolecular order of the ECM is particularly evident in tendon in which millimetre‐long collagen fibrils lie parallel to each other and to the tendon‐long axis (Canty & Kadler 2002). The work in our laboratory is focused on understanding the molecular and cellular basis of collagen fibrillogenesis. The work is directly relevant to understanding the secretion and assembly of large proteins, tissue homeostasis and embryonic development, as well as understanding the aetiology of heritable and acquired diseases of connective tissue including fibrosis.

Materials and methods  A multidisciplinary approach is being used including protein biochemistry, recombinant protein expression, immunofluorescence, immunoEM and serial section reconstruction from electron micrographs.

Results  Using pulse‐chase in organ cultures of embryonic chick tendon we show that approximately 50% of newly synthesized type‐I procollagen is secreted to the ECM where it is cleaved to type‐I collagen, and approximately 50% of the procollagen is cleaved to collagen inside the cells. ImmunoEM shows collagen in specialized secretory vesicles, which bud from the trans‐face of the Golgi apparatus. Serial section reconstruction of E13 chick metatarsal tendon and E15.5 mouse tail tendon shows cross‐striated nascent collagen fibrils, which are approximately 1 µm in length and identical to collagen early collagen fibrils (Graham et al. 2000), enclosed within tube‐like secretory vesicles. The nascent fibrils are secreted via nozzles (plasma membrane protrusions) into tunnel‐shaped spaces between the cells. Bone morphogenetic protein‐1 (ProBMP‐1) is cleaved to active BMP‐1 in the TGN by dibasic convertases (Graham et al. 2000) (e.g. furin) and thereby initiates the assembly of collagen fibrils. Further studies have identified the minimal structure of procollagen C‐proteinase activity of BMP‐1 (Leighton & Kadler 2003).

Discussion  In vitro studies in the 1980s established that collagen fibrillogenesis resembles a crystallization process, in that it proceeds by distinct nucleation and propagation phases. However, there was no obvious mechanism for how the two phases occurred separately in vivo. Our recent studies show that the nucleation phase occurs in specialized secretory vesicles. The early fibrils are deposited in the ECM via nozzles which are long protrusions of the plasma membranes. The propagation phase for fibril assembly occurs in the ECM. Our studies have direct relevance to the assembly of other matrix polymers, e.g. fibrillin and type‐VI collagen, as well as understanding the molecular basis of ECM assembly in diseases such as fibrosis and wound healing.
Decorin in renal inflammation and fibrosis – more than an inhibitor of TGF‐β
Authors: Schaefer Liliana
Published: September 2009

Introduction  Decorin, a small leucine‐rich proteoglycan, participates in extracellular matrix assembly and influences cell behaviour by interacting with signalling membrane receptors and TGF‐β. Treatment with decorin has been shown to have beneficial effects in an acute model of mesangioproliferative glomerulonephritis due to interaction with TGF‐β. The underlying mechanisms, however, remain unclear because upon complex formation with TGF‐β, the cytokine's activity may become increased, decreased or not influenced at all. Hence, the objectives of the present study were twofold: firstly, to provide evidence that decorin influences the course and final outcome of renal inflammation and secondly, to find anti‐fibrotic mechanisms of decorin both related and unrelated to the regulation of TGF‐β activity.

Results  Based on our studies in human diabetic nephropathy, we postulate two regulatory mechanisms by which decorin modulates TGF‐β‐mediated fibrosis: (1) increased quantities of glomerular decorin are synthesized and form complexes with TGF‐β, which then are removed via glomerular capillaries or the urinary tract, and (2) in the presence of type‐I collagen, decorin is able to sequester TGF‐β in the extracellular matrix, thereby withdrawing the cytokine from its cell‐surface receptors. Furthermore, we have compared the evolution of tubulointerstitial fibrosis in wild‐type (WT) and decorin–/– mice in a model of unilateral ureteral obstruction. Without obstruction, kidneys from decorin–/– mice did not differ in any aspect from their WT counterparts. However, already 12 h after obstruction, decorin–/– animals showed lower levels of p27KIP1 and soon thereafter a more pronounced up‐regulation and activation of initiator and effector caspases, followed by enhanced apoptosis of tubular epithelial cells. At later stages, a higher increase of TGF‐β1 became apparent. After 7 days, there was a 15‐fold transient up‐regulation of the related proteoglycan biglycan, which was mainly caused by the appearance of biglycan‐expressing mononuclear cells. Other small proteoglycans showed no similar response. Owing to enhanced degradation of type‐I collagen and increased tubular epithelial cell apoptosis, end‐stage kidneys from decorin–/– animals were more atrophic than WT kidneys.

Conclusion  These data suggest that decorin exerts beneficial effects on renal inflammation, primarily by influencing the expression of a key cyclin‐dependent kinase inhibitor, thereby limiting the degree of apoptosis and tubular atrophy. In later stages, anti‐fibrotic effects of decorin are based on the regulation of TGF‐β1 expression, mononuclear cell infiltration and collagen turnover.
Matrix‐metabolizing enzymes as prime targets of redox regulation by nitric oxide
Authors: Pfeilschifter Josef , Akool El Sayed , Eberhardt Wolfgang
Published: September 2009

Introduction  Dysregulation of extracellular matrix (ECM) turnover is an important feature of many inflammatory processes in the kidney. Rat mesangial cells (MCs) express high levels of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase‐9 (MMP‐9) in response to interleukin‐1β (IL‐1β) (Pfeilschifter & Schwarzenbach 1990; Eberhardt et al. 2000a). The aim of our studies was to elucidate the mechanisms of NO‐dependent modulation of MMP‐9 expression.

Methods  The expression level of MMP‐9 was detected by zymography and by Northern blot analysis, respectively. The influence of NO on mRNA stability was measured by use of actinomycin‐D and by Luciferase reporter gene assays using heterologous MMP‐9 reporter gene constructs. The effects of NO on MMP‐9 transcripts were monitored by in vitro degradation assays and RNA‐binding activity determined by electrophoretic mobility shift assays (EMSA).

Results  MMP‐9 (gelatinase‐B) is a metalloproteinase that is synthesized and secreted in high levels by the glomerular MCs upon treatment with the proinflammatory cytokine IL‐1β mainly via an increase in MMP‐9 transcription (Eberhardt et al. 2000a). The increase in MMP‐9 expression by cytokines is partially due to generation of superoxide and involves the activation of different MAPK pathways (Eberhardt et al. 2000b). Furthermore, we demonstrate that NO substantially inhibits the cytokine‐induced expression of MMP‐9 without affecting MMP‐9 enzyme activity (Eberhardt et al. 2000a). By using actinomycin‐D, we found that NO reduces the half‐life of MMP‐9 mRNA (Akool et al. 2003). The reduction of mRNA stability is attributable to the 3′‐untranslated region of MMP‐9 since introduction of this region confered a negative NO responsiveness to an otherwise nonresponding heterologous MMP‐9 reporter luciferase gene (Akool et al. 2003). By EMSA, we found that cytoplasmic fractions display the consitutive RNA binding of complexes containing the ELAV‐like protein HuR (HuA). Moreover, the RNA binding to three putative AU‐rich elements (AREs) is strongly attenuated by different NO donors (Akool et al. 2003). Correspondingly, the decay of MMP‐9 transcripts was accelerated by cytoplasmic extracts from NO‐treated MCs as shown by in vitro degradation assay (Akool et al. 2003).

Conclusion  NO negatively regulates cytokine‐induced MMP‐9 expression by a post‐transcriptional mechanism which involves inhibition of the mRNA stabilizing ELAV‐like protein HuR.
Emodin ameliorates glucose‐induced fibronectin synthesis in human peritoneal mesothelial cells by inhibiting PKC‐α activation
Authors: Yung Susan , Leung Jack , Tsang Ryan , Liu Zhi Hong , Li Lei Shi , Chan Tak Mao
Published: September 2009

Introduction Peritoneal dialysis (PD) is recognized as an effective treatment for patients with end‐stage renal failure. Conventional PD solutions are unphysiological, as they contain bio‐incompatible concentrations of glucose to provide the osmotic drive (Popovichet al. 1978) Thus, with increasing duration on PD, the peritoneum undergoes progressive structural and functional deterioration. Diabetiform alterations of the peritoneum, for example, the reduplication of the basement membrane, and increased matrix accumulation and deposition within the submesothelium are commonly observed in PD patients. These changes are mediated in part through activation of PKC and induction of TGF‐β1 (Haet al. 2001). Emodin (3‐methyl‐1,6,8 trihydroxyanthraquinone) has previously been demonstrated to reduce cell proliferation and fibronectin synthesis in cultured mesangial cells (Kuoet al. 2001;Yaoet al. 1994). How emodin modulates glucose‐induced fibronectin synthesis in human peritoneal mesothelial cells (HPMCs) has not been elucidated and thus constitutes the aim of this study.

Materials and methods Confluent growth‐arrested HPMCs were cultured under physiological (5 mm) or elevated (30 mm) d‐glucose concentrations in the presence or absence of emodin (20 µg/ml) for periods up to 72 h. PKC‐α activation in HPMCs cultured under control and experimental conditions was determined by Western blot analysis of cytosolic and cell membrane extracts. Synthesis of fibronectin and TGF‐β1 was investigated by a combination of ELISAs, immunohistochemistry and Western blot analysis. Mannitol was used as the hexose control.

Results Exposure of HPMCs to 30 mm d‐glucose resulted in the activation of PKC‐α commencing 12 h after stimulation, as demonstrated by the translocation of PKC‐α from the cytosol to the cell membrane. Maximum induction of PKC‐α was observed 48 h after stimulation and increased 1.4‐fold and 3.7‐fold in the cytosolic and membrane fractions, respectively, compared to 5 mm d‐glucose. Activation of PKC‐α was accompanied by the induction of TGF‐β1 secretion in a time‐dependent manner (3.72 ± 0.29 and 4.30 ± 0.50 pg/µg cellular protein at 24 h and 48 h, respectively, for 30 mm d‐glucose compared to 2.13 ± 0.23 and 2.65 ± 0.32 pg/µg cellular protein at 24 h and 48 h, respectively, for 5 mm d‐glucose,P < 0.001 at both time points) and increased fibronectin synthesis. Whilst emodin had no effect on constitutive PKC‐α, TGF‐β1 or fibronectin synthesis, emodin abrogated elevated glucose stimulation of PKC‐α, and decreased TGF‐β1 secretion and fibronectin synthesis to basal levels.

Discussion Our findings demonstrate that emodin can ameliorate the undesirable effects of concentrated glucose on HPMCs through the suppression of PKC‐α activation. This study suggests that emodin may have therapeutic potential in the prevention or treatment of glucose‐induced structural and functional abnormalities in the peritoneal membrane.
TGF‐β promotes the formation of pyridinoline cross‐links in fibrosis via the induction of LH2 expression
Authors: Slot Annemarie J. Van Der , Zuurmond Anne‐Marie , Abraham David J. , Bank Ruud A.
Published: September 2009

Introduction  The hallmark of fibrosis is an excessive accumulation of collagen, a process in which TGF‐β plays an important role. The deposited collagen shows an increase in pyridinoline cross‐links, due to overhydroxylation of lysine residues within the telopeptides. As we have found that the enzyme responsible for the hydroxylation of the telepeptide lysine residues is lysyl hydroxylase‐2 (LH2), it was examined whether LH2 is increased in fibrotic lesions. TGF‐β is a key mediator in fibrosis, and therefore its effect on the formation of pyridinolines was examined.

Material and methods  Using real‐time PCR, LH2 mRNA expression was measured in fibroblasts cultured from the fibrotic skin of systemic sclerosis (SSc) patients. Furthermore, the amount of pyridinoline cross‐links was analysed in the matrix deposited by fibroblasts stimulated with TGF‐β.

Results  Elevated LH2 mRNA expression levels were found in SSc fibroblasts, which result in increased amounts of pyridinoline cross‐links. Furthermore, increased pyridinoline levels were found in collagen deposited by fibroblasts stimulated with TGF‐β, which was a consequence of increased LH2 mRNA levels.

Conclusion  These data demonstrate for the first time that during fibrotic processes, TGF‐β plays a key role in the formation of pyridinoline cross‐links via the induction of LH2 expression.
Mesangial matrix‐activated mononuclear cells express functional scavenger receptors and accumulate intracellular lipid
Authors: Rahman Enam , Chana Ravinder S. , Ruan Xiong Z. , Powis Stephen H. , Varghese Zac , Wheeler David C.
Published: September 2009

Introduction  Monocyte recruitment into the mesangium and foam cell formation are recognized features of glomerular injury. External signals encountered by these infiltrating cells may determine their behaviour and thereby potentially influence disease outcomes. Our previous studies indicate that activation of monocytes by mesangial matrix stimulates the production of a variety of mediators including inflammatory cytokines and matrix degrading enzymes.

Methods  Using expression of peroxisome proliferator activator receptor‐γ (PPAR‐γ) and scavenger receptor (ScR) as differentiation markers, we examined whether matrix activation was associated with the expression of monocyte characteristics usually associated with a macrophage phenotype. THP‐1 mononuclear cells were incubated for 7 days with 500 µg/ml solublized matrix extracted from cultured human mesangial cells.

Results  Using phorbol methyl ester (PMA) (125 nm) and albumin (500 µg/ml) as positive and negative controls, respectively, we demonstrated that matrix activation of monocytes led to intracellular lipid accumulation as demonstrated by oil red O staining. Matrix activation was also associated with a concentration‐dependent increase in the expression of both ScR and PPAR‐γ mRNA and a corresponding increase in PPAR‐γ protein expression on Western blotting. The presence of functional ScR was confirmed using FACS analysis in which incubation of matrix‐activated monocytes with Dil‐labelled acetylated low‐density lipoprotein (LDL) led to an increase in mean fluorescent intensity of 373% (P < 0.001) as compared to albumin (100%) and PMA (423%). This could be inhibited by addition of excess unlabelled ligand, suggesting specific binding to the ScR. Furthermore, incubation of LDL with mesangial matrix in the absence of cells led to enhanced electrophoretic mobility of recovered lipoprotein on agarose gel. A similar shift was seen when LDL was incubated with Cu2+, a powerful lipoprotein oxidant.

Discussion  These results demonstrate that interactions with mesangial matrix induce expression of monocyte characteristics associated with a macrophage phenotype and promote oxidation of LDL, thereby converting it to an ScR ligand. Such observations may help to explain foam cell formation in the mesangium in the context of glomerular disease.
Domain deletions outside of the catalytic domain of drosophila tolloid result in loss of protease activity
Authors: Canty E.G. , Kadler K.E.
Published: September 2009

Introduction  The tolloid (TLD)‐related zinc metalloproteinases serve multiple functions in development, being involved in both extracellular matrix assembly and TGF‐β signalling. Signalling by bone morphogenetic proteins BMPs, which form a subgroup of the TGF‐β superfamily, is a highly regulated process which involves cleavage of extracellular BMP antagonists by TLD‐like (TLL) proteases. In vertebrates, BMP‐1 and mTLL‐1 cleave the BMP antagonist chordin to release free BMPs, while in invertebrates, TLD cleaves the BMP antagonist short gastrulation (SOG) to release decapentaplegic (DPP). Mammalian TLD (mTLD), TLL‐1 and TLL‐2 share the same domain structure as drosophila TLD, while BMP‐1 is a shorter splice variant of mTLD which lacks the EGF‐2, CUB‐4 and CUB‐5 domains.

Material and methods  Site‐directed mutagenesis was used to produce TLD constructs lacking various domains. These were subcloned into the vector pAc5.1V5His, along with wild‐type TLD and full‐length SOG. Transient transfections in drosophila S2 cells were used to produce recombinant proteins, and aliquots of medium containing the appropriate recombinant proteins were dialysed or desalted as required. In vitro digests of SOG by TLD and the TLD mutants were performed overnight at 25°C in the presence of recombinant DPP (R & D Systems). Digests were analysed by SDS Page and Western blotting, and full‐length SOG and SOG fragments were detected using an anti‐V5 antibody (Invitrogen).

Results  Wild‐type TLD was able to cleave SOG at three distinct sites as previously reported (Marques et al. 1997). A ‘BMP1‐like’ TLD construct, truncated after the CUB3 domain, was efficiently secreted by S2 cells but was unable to cleave SOG. Wild‐type TLD was also found to have enhanced activity in the presence of calcium ions, indicating that it is a calcium‐dependent protease. Removal of the putative calcium‐binding domains from TLD (EGF1 and EGF2) resulted in a decreased amount of enzyme present in the medium of the transfected S2 cells and less (ΔEGF1) or abolished (ΔEGF2 and double deletion) protease activity.

Discussion  These preliminary findings indicate that the EGF‐2, CUB‐4 and CUB‐5 domains are required for cleavage of SOG by TLD. This is in direct contrast to the cleavage of chordin which is actually cleaved most efficiently by the short‐splice variant BMP‐1 (Scott et al. 1999). In vitro cleavage of SOG also requires the presence of DPP, whereas chordin is cleaved in the absence of exogenous BMPs. It is possible that these domains could be involved in interactions with the substrate or alternatively with DPP itself. The putative calcium‐binding domains, EGF1 and EGF2, appear to be required both for secretion and stability of the protein as well as for protease activity. The mechanism whereby calcium augments the protease activity of TLD is unknown, but it could aid the binding of enzyme to substrate or create specific conformational changes in the enzyme to increase activity.
PRG‐4/SZP N‐ and C‐terminal domains: cloning, expression and characterization
Authors: Jones A.R.C. , Hughes C.E. , Wainwright S.D. , Flannery C.R. , Caterson B.
Published: September 2009

Introduction  Proteoglycan‐4 (PRG‐4), also known as superficial zone protein/proteoglycan (SZP), is an approximately 345‐kDa mucinous proteoglycan that has been detected in a variety of tissues including cartilage, tendon, bone, heart and liver (Ikegawa et al. 2000). In the synovial joint, PRG‐4 is specifically synthesized by chondrocytes located in the superficial zone of articular cartilage and by some surface‐lining cells of the synovium (Schumacher et al. 1994). Sequence analyses have shown that the N‐ and C‐terminal vitronectin‐like domains of PRG‐4 may impart interesting functions relevant to synovial joint metabolism (Merberg et al. 1993; Flannery et al. 1999). The objective of this study was to investigate these potential functions, facilitated by the production of PRG‐4 N‐ and C‐terminal domains as recombinant proteins.

Methods  cDNAs for the human N‐terminal (exons 2–5) and bovine C‐terminal (exons 7–12) domains of PRG‐4 were obtained by RT‐PCR and cloned into the expression vector pMT‐BiP for inducible, secreted expression in Drosophila S2 cells. Proteins were purified using FLAG‐M2 antibody affinity chromatography and visualized by SDS‐PAGE and Western blotting with PRG‐4‐specific antibodies. The heparin‐binding properties of recombinant proteins were investigated using heparin affinity chromatography. The interactions of recombinant PRG‐4 domains with human plasminogen activator‐inhibitor (PAI)‐1 and bovine type‐II collagen were assayed using standard ELISA techniques.

Results  Stable cell lines have been generated that express human N‐terminal and bovine C‐terminal PRG‐4 domains. In both cases, two proteins have been purified, possibly due to a splice mechanism by the expression system. N‐terminal sequence data and Western blotting indicate that the two species in each case could represent full‐length and truncated proteins. Analyses of the two PRG‐4 N‐terminal domain species have confirmed the presence of a predicted heparin‐binding domain and indicate that the molecule can bind to PAI‐1, with binding activity localized towards its two somatomedin B domains. The somatomedin B domain of vitronectin is known to bind PAI‐1 (Seiffert 1997). Analyses of the two PRG‐4 C‐terminal species have demonstrated self‐association under nonreducing conditions and binding to heparin and PAI‐1.

Discussion  The exact role of PRG‐4 in the synovial joint is yet to be elucidated. However, these results point towards the interaction of the N‐ and C‐terminal domains of PRG‐4 with structural molecules such as type‐II collagen and heparin, and functional molecules such as PAI‐1, a serpin that is involved in the fibrinolytic cascade and cell adhesion. These properties are in addition to the well‐documented boundary lubricating activity of the central mucinous region of PRG‐4.
Role of the EGF‐like domains in mammalian tolloid (mTLD) secretion and procollagen C‐proteinase activity
Authors: Garrigue‐Antar Laure , Kadler Karl E.
Published: September 2009

Introduction  Bone morphogenetic protein (BMP)‐1 and its larger splice variant mammalian tolloid (mTLD) belong to the tolloid group of astacin‐like metalloproteinases that are fundamental to tissue patterning and extracellular matrix assembly. BMP‐1 and mTLD exhibit similar substrate specificity in vitro; however, BMP‐1 is a much better procollagen C‐proteinase than mTLD. mTLD consists of a prodomain (which is cleaved by a furin‐like enzyme) (Leighton & Kadler 2003), a zinc metalloproteinase domain and a C‐terminal part comprising five CUB domains thought to be important for protein–protein interactions (Hartigan et al. 2003), and two EGF‐like domains, which in other proteins are involved in calcium ion binding. BMP‐1 lacks the most C‐terminal two CUB domains and one EGF‐like domain. mTLD activity is known to be calcium ion dependent, as demonstrated for the chick homologue (Hojima et al. 1985). In our current work, we are studying the role of the EGF‐like domains in the secretion and procollagen C‐proteinase activity of mTLD, and the contribution that these domains made to calcium ion dependency.

Materials and methods  We designed proteins lacking EGF1, EGF2 or both. NotI sites were introduced by PCR at the borders of the EGF domain of a cDNA clone encoding a V5‐His mTLD. Restriction enzyme digestion was used to delete individual domains. The mutant constructs in pCEP4 were stably transfected into 293‐EBNA cells. Expression was analysed by Western blot. The wild‐type and the mutant enzymes were purified on a nickel ion column, and their activity was determined by cleavage of type‐I procollagen in the presence or absence of 5 mm CaCl2.

Results  We showed that (1) the mTLD proteins lacking EGF1, EGF2 or EGF1 + EGF2 were poorly secreted into the culture medium compared to mTLD and (2) the EGF deletion mutants remained calcium ion dependent, but some differences were seen. Most notably, the ΔEGF2 and ΔEGF1 + ΔEGF2 mutants were found to be better C‐proteinases than the wild‐type enzyme in the presence of calcium ions.

Conclusion  From these preliminary data, we concluded that (1) the EGF domains are necessary for efficient secretion (2) both EGF1 and EGF2 domains contribute to the calcium ion dependency of mTLD and (3) the EGF2 domain might be a Ca2+‐activated hinge that ‘swings’ the CUB‐4 and CUB‐5 domains away from the active site. The ?EGF2 mTLD might be expected to have an open conformation, thereby making it a better C‐proteinase than the wild‐type enzyme, and (?4) Ca2+ ions are bound by other domains in mTLD and not only by the EGF‐like domains.
Age‐related changes in the glycosaminoglycans of human meniscal aggrecan
Authors: Al‐Jafary Meneerah , Huckerby Thomas N. , Lauder Robert M.
Published: September 2009

Introduction  Meniscal damage and degradation, which are strongly correlated with subsequent OA, have been identified in approximately 60% of people over 60 years of age. Age‐related changes in articular cartilage glycosaminoglycans (GAGs) have been described, and used to facilitate the study of pathology‐related changes (Plass et al. 1998). However, such data do not yet exist for the meniscus.

Materials and methods  Undamaged human menisci were obtained following leg amputations, and the vascular and avascular zones of each lateral and medial meniscus were extracted into 4 m GuHCl. Aggrecan was recovered in the A1 fraction following CsCl density gradient centrifugation, and the relative abundance of chondroitin, dermatan and keratan sulphates (CS, DS and KS) was examined by NMR spectroscopy at 400 MHz and 43 °C.

Results  Human meniscal aggrecan was shown to contain CS, DS and KS, and our data show age‐related changes in the relative abundance of these GAGs. The change was similar for medial and lateral menisci and for the vascular and avascular zones within these.

The KS abundance in aggrecan from young menisci (<15 years) was found to be 15–20% of the total GAGs. However, in older samples, it comprised only 7–12% of the GAGs.

We have confirmed the presence of DS in human meniscal aggrecan and show that the abundance of DS gradually falls from approximately 16% at 10 years to 2–4% at 75 years. There is some variability between humans, although the trend is clear and for each human there is good agreement between medial and lateral menisci and vascular and avascular locations.

The levels of CS comprise the remainder of the GAG attached to aggrecan and contribute the remainder of the GAG abundance. This can be seen to increase from 67 to 72% at 10 years to approximately 90% at 75 years.

Discussion  Our data show a clear age‐related change in the relative abundance CS, DS and KS from human meniscal aggrecan. The data show a decrease in the abundance of KS and DS and a concomitant increase in CS levels. These observations differ from those widely seen for articular cartilage, in which the levels of CS are seen to fall with age.

We have confirmed that DS is a component of human meniscal aggrecan in agreement with previous work (McNicol & Roughley 1980). However, previously reported levels of DS, approximately 20%, are those found only in younger menisci.

Absolute levels of these GAGs have not yet been determined, and hence the mechanisms which bring about this relative increase in CS with age may include either changes in biosynthetic output and/or widespread GAG loss in which KS and DS loss increases with age.
Systematic review: endoscopic and imaging‐based techniques in the assessment of portal haemodynamics and the risk of variceal bleeding
Authors: SGOUROS S. N. , VASILIADIS K. V. , PEREIRA S. P.
Published: September 2009

Background  Invasive measurement of the hepatic venous pressure gradient (HVPG) is regarded as the gold standard for risk stratification and the evaluation of pharmaceutical agents in patients with portal hypertension.

Aim  To review the techniques for endoscopic and imaging‐based assessment of portal haemodynamics, with particular emphasis on trials where the results were compared with HVPG or direct portal pressure measurement.

Methods  Systematic search of the MEDLINE electronic database with keywords: portal hypertension, variceal bleeding, variceal pressure, endoscopic ultrasound, Doppler ultrasonography, magnetic resonance angiography, CT angiography, hepatic venous pressure gradient.

Results  Computed tomography angiography and endoscopic ultrasound (EUS) have been both employed for the diagnosis of complications of portal hypertension and for the evaluation of the efficacy of endoscopic therapy. Colour Doppler ultrasonography and magnetic resonance angiography has given discrepant results. Endoscopic variceal pressure measurements either alone or combined with simultaneous EUS, correlate well with HVPG and risk of variceal bleeding and have a low interobserver variability.

Conclusions  Endoscopic and imaging‐based measurements of portal haemodynamics provide an alternate means for the assessment of complications of portal hypertension. Further studies are required to validate their use in risk stratification and the evaluation of drug therapies in patients with portal hypertension.
The allogeneic graft‐versus‐cancer effect
Authors: Ringdén Olle , Karlsson Helen , Olsson Richard , Omazic Brigitta , Uhlin Michael
Published: September 2009

Allogeneic haematological stem cell transplantation (HSCT) has developed into immunotherapy. Donor CD4+, CD8+and natural killer (NK) cells have been reported to mediate graft‐versus‐leukaemia (GVL) effects, using Fas‐dependent killing and perforin degranulation to eradicate malignant cells. Cytokines, such as interleukin‐2, interferon‐γ and tumour necrosis factor‐α potentiate the GVL effect. Post‐transplant adoptive therapy of cytotoxic T‐cells (CTL) against leukaemia‐specific antigens, minor histocompatibility antigens, or T‐cell receptor genes may constitute successful approaches to induce anti‐tumour effects. Clinically, a significant GVL effect is induced by chronic rather than acute graft‐versus‐host disease (GVHD). An anti‐tumour effect has also been reported for myeloma, lymphoma and solid tumours. Reduced intensity conditioning enables HSCT in older and disabled patients and relies on the graft‐versus‐tumour effect. Donor lymphocyte infusions promote the GVL effect and can be given as escalating doses with response monitored by minimal residual disease. A high CD34+cell dose of peripheral blood stem cells increases GVL. There is a balance between effective immunosuppression, low incidence of GVHD and relapse. For instance, T‐cell depletion of the graft increases the risk of relapse. This paper reviews the current knowledge in graft‐versus‐cancer effects. Future directions, such as immunotherapy using leukaemia‐specific CTLs, allo‐depleted T‐cells and suicide gene manipulated T‐cells, are presented.
Allergic sensitization is enhanced in early life through toll‐like receptor 7 activation
Authors: Phipps S. , Hansbro N. , Lam C. E. , Foo S. Y. , Matthaei K. I. , Foster P. S.
Published: September 2009

Background
Prospective cohort studies suggest that children hospitalized in early life with severe infections are significantly more likely to develop recurrent wheezing and asthma.

Objective
Using an inhalational mouse model of allergic airways inflammation, we sought to determine the effect of viral and bacterial‐associated molecular patterns on the magnitude of the allergic inflammatory response and whether this effect was age dependent.

Methods
BALB/c mice were sensitized by intranasal administration of endotoxinlow ovalbumin (OVA) in the absence or presence of viral single‐stranded (ss)RNA, lipoteichoic acid or flagellin as neonates (within the first 24 h of life) or as weanlings (4 weeks of age). Mice were challenged four times with OVA at 6 weeks of age and end‐points (bronchoalveolar lavage cytology, histology, antigen‐specific T and B cell responses) determined at 7 weeks of age.

Results
Inhalational sensitization (<24 h or 4 weeks of age) and challenge with OVA induced a mild allergic inflammatory response in the airways as indicated by increased numbers of eosinophils and mucus cells, elevated serum OVA‐specific IgG1, and production of T helper 2 (Th2) cytokines. Mice sensitized to endotoxinlow OVA at birth in the presence of ssRNA or lipoteichoic acid, but not flagellin, showed an increase in the numbers of airway and tissue eosinophils, mucus producing cells and antigen‐specific production of IL‐13 as compared with mice exposed only to endotoxinlow OVA. By contrast, all three TLR ligands failed to increase the magnitude of OVA‐induced allergic inflammation in mice sensitized as weanlings.

Conclusions
Recognition of distinct microbial‐associated patterns in early life may preferentially promote the de novo differentiation of bystander, antigen‐specific CD4+ T cells toward a Th2 phenotype, and promote an asthma‐like phenotype upon cognate antigen exposure in later life.
Reliability and validity of the caregiver quality of life index‐cancer (CQOLC) scale in Turkish cancer caregivers
Authors: Bektas Hicran A , Ozer Zeynep C
Published: September 2009

Aims and objectives.  The aim of this study was to translate the Caregiver Quality of Life Index‐Cancer into Turkish and test the reliability and validity of the Turkish version of the Caregiver Quality of Life Index‐Cancer in Turkey.

Background.  Cancer is a chronic illness that affects the entire family and deteriorates the quality of life of cancer caregivers. The Caregiver Quality of Life Index‐Cancer is a widely used instrument to measure quality of life in cancer caregivers. Therefore, identifying and increasing quality of life benefits of cancer caregivers.

Design.  Methodological research design.

Method.  This study describes the translation process of The Caregiver Quality of Life Index‐Cancer into Turkish and testing its reliability and validity on quality of life in cancer caregivers. The questionnaire was translated using a back‐translation technique. The statistical analysis was carried out using Cronbach’s alpha to test the internal consistency of the Caregiver Quality of Life Index‐Cancer scale while confirmatory and exploratory factor analyses were carried out using principal component analysis with varimax rotation and Kaiser Normalization to test its construct validity.

Results.  The Cronbach’s alpha was found to be reliability for the total scale was 0·88 and subscale alpha coefficients ranged from 0·73–0·83. Confirmatory factor analysis resulted in four factor structure: burden, distruptiveness, positive adaptation and financial concerns.

Conclusion.  The effects of providing care for patients with cancer on caregiver quality of life have not yet been adequately explored in Turkey. The results of this study suggest that the Turkish version of the Caregiver Quality of Life Index‐Cancer is a reliable and valid supplementary measure of the quality of life in cancer caregivers in Turkey.

Relevance of clinical practice.  In research and practice, valid measurement instruments are needed to assess Quality of Life in Turkish cancer caregivers. The Caregiver Quality of Life Index‐Cancer scale is simple to administer and nurses by using this equipment in routine appointments will be able to better identify caregiver at risk for developing cancer related distress and worse Quality of Life.
Relationship between call light use and response time and inpatient falls in acute care settings
Authors: Tzeng Huey‐Ming , Yin Chang‐Yi
Published: September 2009

Aims and objectives.  This exploratory study used archived hospital data to determine whether the call light use rate and the average call light response time contribute to the fall and the injurious fall rates in acute care settings.

Background.  Inpatients often use call lights to seek nurses’ attention and assistance. Although implied in patient safety, no studies have examined data related to the call light use or the response time to call lights collected via existing tracking mechanisms to monitor nursing practice.

Design.  The study was conducted in a Michigan community hospital and used archived hospital data for analyses for the period from February 2007–June 2008. The unit of analysis was unit‐week.

Method.  The call light use rate per patient‐day was calculated based on information retrieved from the call light tracking system. The average response time in seconds was used as generated from the tracking system. The fall and injurious fall rates per 1000 patient‐days were calculated based on the fall incident reports. spss was used for data analyses. One‐way anova and correlation analyses were conducted.

Results.  More calls for assistance related to less fall‐related patient harm. Surprisingly, longer response time to call lights also related to fewer total falls and less fall‐related patient harm. Generally speaking, more call light use related to longer response times.

Conclusions.  This study’s findings challenged the appropriateness of targeting the goals of reducing the frequency of call light use and the fall rates as two outcome indicators of conducting hourly patient rounds.

Relevance to clinical practice.  Encouraging call light use is a key to reducing injurious fall rates. Unit managers should routinely monitor the trend of the call light use rate and ensure that the call light use rate is maintained at least above the mean rate.