bluuebunny/crossref_metadata_2025 huggingface dataset:
On exploiting hierarchical label structure with pairwise classifiers
Authors: Fürnkranz Johannes , Sima Jan Frederik
Published: April 2011

The goal of this work was to test whether the performance of a regular pairwise classifier can be improved when additional information about the hierarchical class structure is added to the training sets. Somewhat surprisingly, the additional information seems to hurt the performance. We explain this with the fact that the structure of the class hierarchy is not reflected in the distribution of the instances.
Unexpected results in automatic list extraction on the web
Authors: Weninger Tim , Fumarola Fabio , Barber Rick , Han Jiawei , Malerba Donato
Published: April 2011

The discovery and extraction of general lists on the Web continues to be an important problem facing theWeb mining community. There have been numerous studies that claim to automatically extract structured data (i.e. lists, record sets, tables, etc.) from the Web for various purposes. Our own recent experiences have shown that the list-finding methods used as part of these larger frameworks do not generalize well and therefore ought to be reevaluated. This paper briefly describes some of the current approaches, and tests them on various list-pages. Based on our findings, we conclude that analyzing aWeb page's DOM-structure is not sufficient for the general list finding task.
Knowledge discovery from sensor data (SensorKDD)
Authors: Chandola Varun , Omitaomu Olufemi A. , Ganguly Auroop R. , Vatsavai Ranga R. , Chawla Nitesh V. , Gama Joao , Gaber Mohamed M.
Published: April 2011

Sensor data is being collected at an unprecedented rate across a variety of domains from a broad spectrum of sources, such as wide-area sensor infrastructures, remote sensing instruments, RFIDs, and wireless sensor networks. With the recent proliferation of smart-phones, and similar GPS enabled mobile devices, collection of sensor data is no longer limited to scientific communities, but has reached general public. With massive volumes of such disparate, dynamic, and geographically distributed data available, many high-priority applications have been identified that involve analysis of such data to solve real world problems such as understanding climate change and its impacts, electric grid monitoring, disaster preparedness and management, national or homeland security, and the management of critical infrastructures.

Given the unique characteristics of sensor data, particularly its spatiotemporal nature and presence of constraints associated with the data collection and computational resources, there have been many research efforts to analyze the sensor data which build upon the general research in the data mining community but are significantly different in terms of how they address the specific challenges encountered when dealing with sensor data. In particular, the raw data from sensors needs to be efficiently managed and transformed to usable information through data fusion, which in turn must be converted to predictive insights via knowledge discovery, ultimately facilitating automated or humaninduced tactical decisions or strategic policy based on decision sciences and decision support systems.

Keeping in view the requirements of the emerging field of knowledge discovery from sensor data, we took initiative to develop a community of researchers with common interests and scientific goals, which culminated into the organization of SensorKDD series of workshops in conjunction with the prestigious ACM SIGKDD International Conference of Knowledge Discovery and Data Mining. In this report, we summarize events at the Fourth ACM-SIGKDD International Workshop on Knowledge Discovery form Sensor Data (SensorKDD 2010).
Amygdala activation by corticosterone alters visceral and somatic pain in cycling female rats
Authors: Gustafsson Jenny K. , Greenwood-Van Meerveld Beverley
Published: April 2011

Irritable bowel syndrome (IBS) is often seen in women, and symptom severity is known to vary over the menstrual cycle. In addition, activation of the hypothalamic-pituitary-adrenal (HPA) axis enhances symptomology and patients with IBS have increased activation of the amygdala, a brain region known to facilitate HPA output. However, little is known about the effects of amygdala activation during different stages of the menstrual cycle. We therefore investigated the effects of amygdala activation on somatic and visceral pain perception over the rat estrous cycle. Female Wistar rats were implanted with either corticosterone (Cort) or cholesterol as a control onto the dorsal margin of the central amygdala. Visceral sensitivity was quantified by recording the visceromotor response (VMR) to colorectal distension (CRD) and somatic sensitivity was assessed via the Von Frey test. In cholesterol controls, both visceral and somatic sensitivity varied over the estrous cycle. Rats in proestrus/estrus responded to CRD with an increased VMR compared with rats in metestrus/diestrus. Somatic sensitivity followed a similar pattern with enhanced sensitivity during proestrus/estrus compared with metestrus/diestrus. Elevated amygdala Cort induced visceral hypersensitivity during metestrus/diestrus but had no effect during proestrus/estrus. In contrast, elevated amygdala Cort increased somatic sensitivity during both metestrus/diestrus and proestrus/estrous. These results suggests that amygdala activation by Cort eliminates spontaneously occurring differences in visceral and somatic pain perception, which could explain the lowered pain thresholds and higher incidence of somatic pain observed in women with IBS.
Dietary fiber enhances TGF-β signaling and growth inhibition in the gut
Authors: Cao Yanna , Gao Xuxia , Zhang Weili , Zhang Guohua , Nguyen Anthony K. , Liu Xianghua , Jimenez Fernando , Cox Charles S. , Townsend Courtney M. , Ko Tien C.
Published: April 2011

Dietary fiber intake links to decreased risk of colorectal cancers. The underlying mechanisms remain unclear. Recently, we found that butyrate, a short-chain fatty acid produced in gut by bacterial fermentation of dietary fiber, enhances TGF-β signaling in rat intestinal epithelial cells (RIE-1). Furthermore, TGF-β represses inhibitors of differentiation (Ids), leading to apoptosis. We hypothesized that dietary fiber enhances TGF-β's growth inhibitory effects on gut epithelium via inhibition of Id2. In this study, Balb/c and DBA/2N mice were fed with a regular rodent chow or supplemented with a dietary fiber (20% pectin) and Smad3 level in gut epithelium was measured. In vitro, RIE-1 cells were treated with butyrate and TGF-β1, and cell functions were evaluated. Furthermore, the role of Ids in butyrate- and TGF-β-induced growth inhibition was investigated. We found that pectin feeding increased Smad3 protein levels in the jejunum (1.47 ± 0.26-fold, P = 0.045, in Balb/c mice; 1.49 ± 0.19-fold, P = 0.016, in DBA/2N mice), and phospho-Smad3 levels (1.92 ± 0.27-fold, P = 0.009, in Balb/c mice; 1.83 ± 0.28-fold, P = 0.022, in DBA/2N mice). Butyrate or TGF-β alone inhibited cell growth and induced cell cycle arrest. The combined treatment of butyrate and TGF-β synergistically induced cell cycle arrest and apoptosis in RIE-1 cells and repressed Id2 and Id3 levels. Furthermore, knockdown of Id2 gene expression by use of small interfering RNA caused cell cycle arrest and apoptosis. We conclude that dietary fiber pectin enhanced Smad3 expression and activation in the gut. Butyrate and TGF-β induced cell cycle arrest and apoptosis, which may be mediated by repression of Id2. Our results implicate a novel mechanism of dietary fiber in reducing the risk of colorectal cancer development.
Skin surface cooling improves orthostatic tolerance following prolonged head-down bed rest
Authors: Keller David M. , Low David A. , Davis Scott L. , Hastings Jeff , Crandall Craig G.
Published: April 2011

Prolonged exposure to microgravity, as well as its ground-based analog, head-down bed rest (HDBR), reduces orthostatic tolerance in humans. While skin surface cooling improves orthostatic tolerance, it remains unknown whether this could be an effective countermeasure to preserve orthostatic tolerance following HDBR. We therefore tested the hypothesis that skin surface cooling improves orthostatic tolerance after prolonged HDBR. Eight subjects (six men and two women) participated in the investigation. Orthostatic tolerance was determined using a progressive lower-body negative pressure (LBNP) tolerance test before HDBR during normothermic conditions and on day 16 or day 18 of 6° HDBR during normothermic and skin surface cooling conditions (randomized order post-HDBR). The thermal conditions were achieved by perfusing water (normothermia ∼34°C and skin surface cooling ∼12–15°C) through a tube-lined suit worn by each subject. Tolerance tests were performed after ∼30 min of the respective thermal stimulus. A cumulative stress index (CSI; mmHg LBNP·min) was determined for each LBNP protocol by summing the product of the applied negative pressure and the duration of LBNP at each stage. HDBR reduced normothermic orthostatic tolerance as indexed by a reduction in the CSI from 1,037 ± 96 mmHg·min to 574 ± 63 mmHg·min ( P < 0.05). After HDBR, skin surface cooling increased orthostatic tolerance (797 ± 77 mmHg·min) compared with normothermia ( P < 0.05). While the reduction in orthostatic tolerance following prolonged HDBR was not completely reversed by acute skin surface cooling, the identified improvements may serve as an important and effective countermeasure for individuals exposed to microgravity, as well as immobilized and bed-stricken individuals.
miR-15 and miR-16 Are Direct Transcriptional Targets of E2F1 that Limit E2F-Induced Proliferation by Targeting Cyclin E
Authors: Ofir Matan , Hacohen Dalia , Ginsberg Doron
Published: April 2011

microRNAs (miR) are small noncoding RNA molecules that have recently emerged as critical regulators of gene expression and are often deregulated in cancer. In particular, miRs encoded by the miR-15a, miR-16-1 cluster seem to act as tumor suppressors. Here, we evidence that the miR-15a, miR-16-1 cluster and related miR-15b, miR-16-2 cluster comprise miRs regulated by E2F1, a pivotal transcription factor that can induce both proliferation and cell death. E2F1 is a critical downstream target of the tumor suppressor retinoblastoma (RB). The RB pathway is often inactivated in human tumors resulting in deregulated E2F activity. We show that expression levels of the 4 mature miRs, miR-15a, miR-16-1 and miR-15b, miR-16-2, as well as their precursor pri-miRNAs, are elevated upon activation of ectopic E2F1. Moreover, activation of endogenous E2Fs upregulates expression of these miRs and endogenous E2F1 binds their respective promoters. Importantly, we corroborate that miR-15a/b inhibits expression of cyclin E, the latter a key direct transcriptional target of E2F pivotal for the G1/S transition, raising the possibility that E2F1, miR-15, and cyclin E constitute a feed-forward loop that modulates E2F activity and cell-cycle progression. In support of this, ectopic expression of miR-15 inhibits the G1/S transition, and, conversely, inhibition of miR-15 expression enhances E2F1-induced upregulation of cyclin E1 levels. Furthermore, inhibition of both miR-15 and miR-16 enhances E2F1-induced G1/S transition. In summary, our data identify the miR-15 and miR-16 families as novel transcriptional targets of E2F, which, in turn, modulates E2F activity. Mol Cancer Res; 9(4); 440–7. ©2011 AACR.
The French Society of Neonatology’s Proposals for Neonatal End-of-Life Decision-Making
Authors: Dageville C. , Bétrémieux P. , Gold F. , Simeoni U. ,   for the Working Group on Ethical Issues in Perinatology1
Published: April 2011

Background: Opinions and practice regarding end-of-life decisions in neonatal medicine show considerable variations between countries. A recent change of the legal framework, together with an ongoing debate among French neonatologists, led the French Society of Neonatology to reconsider and update its previous recommendations. Objectives: To propose a set of recommendations on the ethical principles to be respected in the making and application of end-of-life decisions. Methods: A multidisciplinary working group on ethical issues in perinatal medicine composed of neonatologists, obstetricians and ethicists. Results: Withholding or withdrawing life-sustaining treatment may be acceptable, and unreasonable therapeutic obstinacy is condemned. This implies that the child’s best interests must always be the central consideration. Although the parents must be involved in the decision process so that they form an alliance with the healthcare team, and a collegial approach is of utmost importance, any crucial decision affecting the patient’s life calls for individual medical responsibility. Because every newborn is rightfully an integral member of a human family, his or her dignity must be preserved. The goal of palliative care is to preserve the quality of a life, also at its end. The intention underlying an act has to be analyzed perceptively. Euthanasia, i.e. to perform an act with the deliberate intention to cause or hasten a patient’s death, is legally and morally forbidden. Conversely, to withhold or withdraw a life-sustaining treatment can be justified when the intention is to cease opposing, in an unreasonable manner, the natural course of a disease. Conclusions: This statement provides the principles identified by French neonatologists on which to base their decisions concerning the ending of life. Arguments are set forth, discussed and compared with international statements and previously published considerations.
A Case of γ-Butyrolactone Associated with Severe Withdrawal Delirium and Acute Renal Failure
Authors: Bhattacharya Indrani S. , Watson Fiona , Bruce Malcolm
Published: April 2011

γ-Butyrolactone (GBL) is a popular drug of abuse which is easily available over the internet. Following a UK classification change to a class C drug in January 2010, internet supply has become difficult. Some of the effects have resulted in sourcing GBL from industrial solvents. We report a case of a 24-year-old man who was admitted for detoxification from GBL. He reported having sourced the GBL by diluting the contents of nail varnish remover pads with water. During his admission he developed a severe withdrawal delirium and acute renal failure. He required admission to the intensive care unit. Physicians and psychiatrists should be aware of toxic sources of GBL leading to renal failure and consider GBL in those presenting with agitation, psychosis or coma.
Hypersensitivity Pneumonitis Associated with Inhalation of Catechin-Rich Green Tea Extracts
Authors: Otera Hiroshi , Tada Kimihide , Sakurai Toshiyasu , Hashimoto Kimio , Ikeda Akihiko
Published: April 2011

A 51-year-old man presented with fever and fatigue after 3.5 months of antituberculosis therapy. High-resolution computed tomography of his chest revealed new ground-glass opacities and poorly defined centrilobular nodules. He had undergone catechin inhalation for 1 month. We diagnosed hypersensitivity pneumonitis (HP) based on the clinical course, bronchoscopy and a challenge test. Cases of HP due to inhalation of extracted catechin powder are rare. Although it has many known positive attributes, it is necessary to be aware that catechin can cause HP.
A Seven-Year Survey of Management of Coagulase-Negative Staphylococcal Sepsis in the Neonatal Intensive Care Unit: Vancomycin May Not Be Necessary as Empiric Therapy
Authors: Hemels Marieke A.C. , van den Hoogen Agnes , Verboon-Maciolek Malgorzata A. , Fleer André , Krediet Tannette G.
Published: April 2011

Background: The typical empiric therapy for coagulase-negative staphylococcal (CONS) sepsis includes vancomycin. In our neonatal intensive care unit, we have consistently avoided the use of vancomycin to treat CONS sepsis, except for specific cases, and have used instead cefazolin as empiric agent. Objectives: The clinical outcome of infants with CONS sepsis was evaluated in relation to the susceptibility of CONS blood isolates to cefazolin over a period of 7 years. Methods: Clinical characteristics, symptoms of sepsis and antibiotic use were studied retrospectively. Susceptibility of CONS blood isolates to cefazolin was determined by E-test. Results: Of 163 infants with proven CONS sepsis, 121/140 (86%) infants with a cefazolin-susceptible (minimum inhibition concentration (MIC) ≤8 mg/l) and 21/23 (91%) with a cefazolin-resistant (MIC ≧32 mg/l) blood isolate were treated with cefazolin. 21 (13%) infants were switched to vancomycin, in only 3 of them CONS had become resistant to cefazolin. The majority (81%) of the infants with a good response to cefazolin had the indwelling central venous catheter removed, in contrast to only 22% of the infants with cefazolin treatment failure. Median cefazolin MIC values were 0.75–2 mg/l during the study period. Conclusions: The great majority of infants with CONS sepsis was successfully treated with cefazolin. The use of vancomycin could be restricted to specific cases. Despite the consistent use of cefazolin in neonatal CONS sepsis over an extended period of time, cefazolin MIC values remained low and in the susceptible range. Removal of the central venous catheter in infants with clinical symptoms of sepsis is an important therapeutic measure.
Deafferentation of the Affected Arm
Authors: Weiss Thomas , Sens Elisabeth , Teschner Ulrike , Meissner Winfried , Preul Christoph , Witte Otto W. , Miltner Wolfgang H.R.
Published: April 2011

Background and Purpose—

Reduced somatosensation is a common impairment after stroke. This somatosensory deficit is known to be a reliable predictor of poor rehabilitation outcome. Several methods of physical therapy have addressed this problem, but with only moderate success. Here, we used a new neural plasticity-based approach, ie, a simple, inexpensive, pharmacologically induced temporary functional deafferentation (TFD) of the forearm to investigate whether TFD might result in beneficial effects on the somatosensory sensibility and motor capacity of the stroke-affected hand.

Methods—

Examination was performed over 2 consecutive days of an efficient rehabilitation program for stroke patients referred to as constraint-induced movement therapy. Patients were deafferented on one of these days but not on the other (placebo session). The order of deafferentation and nondeafferentation was counterbalanced across patients. TFD of the stroke-affected forearm was realized using an anesthetic cream. Somatosensory abilities were assessed by a Grating orienting task, and a shape-sorter drum task was used to test motor performance. Both tests were performed each day before and after the constraint-induced movement therapy training session.

Results—

We found significantly better outcomes for Grating orienting task and shape-sorter drum task after TFD on the forearm as compared to placebo, indicating increased somatosensory abilities and motor performance in stroke patients using the simple TFD procedure.

Conclusions—

The improvement was achieved during the course of one of the best established poststroke rehabilitation programs, suggesting that TFD on the more affected forearm might become an efficient additional tool in stroke rehabilitation.
Effects of a Single Dose of Dantrolene in Patients With Cerebral Vasospasm After Subarachnoid Hemorrhage
Authors: Muehlschlegel Susanne , Rordorf Guy , Sims John
Published: April 2011

Background and Purpose—

New therapies for cerebral vasospasm after subarachnoid hemorrhage are needed because of its high morbidity and mortality rates. We investigated the feasibility and safety of a single dose of intravenous dantrolene and its effect on transcranial Doppler in cerebral vasospasm after subarachnoid hemorrhage.

Methods—

In a prospective, open-label, single-dose ascending safety trial, 5 patients received intravenous dantrolene 1.25 mg/kg and the next 5 patients received 2.5 mg/kg over the course of 60 minutes. All other infusions were kept steady and hemodynamic parameters were recorded. Transcranial Doppler was performed at 0, 45, 90, and 135 minutes relative to infusion start. Basic chemistries, serum osmolality, arterial blood gas, and liver enzymes were measured before and after.

Results—

Laboratory values and hemodynamic parameters remained unchanged except for a decrease in the systolic blood pressure in the low-dose group (−8 mm Hg; 95% CI, −26 to 10 mm Hg;
P
=0.027). After correcting for this decrease in blood pressure, peak systolic transcranial Doppler velocities decreased significantly (−26 cm/s; 95% CI, −47 to −5 cm/s;
P
=0.02), with a borderline change in mean velocities in the low-dose group (−16 cm/s; 95% CI, −36 to 4 cm/s;
P
=0.07) and peak systolic transcranial Doppler velocity in the high-dose group (−26 cm/s; 95% CI, −56 to 5 cm/s;
P
=0.05).

Conclusions—

In this pilot study, a single dose of intravenous dantrolene in cerebral vasospasm after subarachnoid hemorrhage appears feasible while inhibiting vasoconstriction in the low-dose group, but it may lower blood pressure. Our study provides useful data for the design of larger future studies.

Clinical Trial Registration—

URL:
http://clinicaltrials.gov
. Unique identifier: NCT00964548.
Late Reopening of Adequately Coiled Intracranial Aneurysms
Authors: Ferns Sandra P. , Sprengers Marieke E.S. , van Rooij Willem Jan , van Zwam Wim H. , de Kort Gérard A.P. , Velthuis Birgitta K. , Schaafsma Joanna D. , van den Berg René , Sluzewski Menno , Brouwer Patrick A. , Rinkel Gabriël J.E. , Majoie Charles B.L.M.
Published: April 2011

Background and Purpose—

In aneurysms that are adequately occluded 6 months after coiling, the risk of late reopening is largely unknown. We assessed the occurrence of late aneurysm reopening and possible risk factors.

Methods—

From January 1995 to June 2005, 1808 intracranial aneurysms were coiled in 1675 patients at 7 medical centers. At 6 months, 1066 aneurysms in 971 patients were adequately occluded. At mean 6.0 years after coiling, of the 971 patients, 400 patients with 440 aneurysms underwent 3 Tesla magnetic resonance angiography to assess occlusion status of the aneurysms. Proportions and corresponding 95% CI of aneurysm reopening and retreatment were calculated. Risk factors for late reopening were assessed by univariate and multivariate logistic regression analysis, and included patient sex, rupture status of aneurysms, aneurysm size ≥10 mm, and aneurysm location.

Results—

In 11 of 400 patients (2.8%; 95% CI, 1.4–4.9%) with 440 aneurysms (2.5%; 95% CI, 1.0–4.0%), late reopening had occurred; 3 reopened aneurysms were retreated (0.7%; 95% CI, 0.2–1.5%). Independent predictors for late reopening were aneurysm size ≥10 mm (OR 4.7; 95% CI, 1.3–16.3) and location on basilar tip (OR 3.9; 95% CI, 1.1–14.6). There were no late reopenings in the 143 anterior cerebral artery aneurysms.

Conclusions—

For the vast majority of adequately occluded intracranial aneurysms 6 months after coiling (those <10 mm and not located on basilar tip), prolonged imaging follow-up within the first 5 to 10 years after coiling does not seem beneficial in terms of detecting reopened aneurysms that need retreatment. Whether patients might benefit from screening beyond the 5- to 10-year interval is not yet clear.
Formulating the Rasch Differential Item Functioning Model Under the Marginal Maximum Likelihood Estimation Context and Its Comparison With Mantel–Haenszel Procedure in Short Test and Small Sample Conditions
Authors: Paek Insu , Wilson Mark
Published: April 2011

This study elaborates the Rasch differential item functioning (DIF) model formulation under the marginal maximum likelihood estimation context. Also, the Rasch DIF model performance was examined and compared with the Mantel–Haenszel (MH) procedure in small sample and short test length conditions through simulations. The theoretically known relationship of the DIF estimators between the Rasch DIF model and the MH procedure was confirmed. In general, the MH method showed a conservative tendency for DIF detection rates compared with the Rasch DIF model approach. When there is DIF, the z test (when the standard error of the DIF estimator is estimated properly) and the likelihood ratio test in the Rasch DIF model approach showed higher DIF detection rates than the MH chi-square test for sample sizes of 100 to 300 per group and test lengths ranging from 4 to 39. In addition, this study discusses proposed Rasch DIF classification rules that accommodate statistical inference on the direction of DIF.
Distribution of IκB Proteins in Gastric Mucosa and Other Organs of Mouse and Gerbil
Authors: Koyama Masamichi , Spicer Samuel S. , Schulte Bradley A.
Published: April 2011

The NF-κB/IκB complex is a major transcription regulator of inflammatory and immune responses. Helicobacter pylori infection causes chronic inflammation in gastric mucosa by inducing dissociation of the inhibitory IκB protein from the complex with a resulting increased expression of interleukin (IL)-8. To clarify which of several known IκB proteins could be involved in this inflammatory response, we undertook immunohistochemical examination of normal mouse stomach as well as other murine tissues for comparison, using polyclonal antibodies specific for α-, β-, γ-, and ∊-isoforms of IκB. The results showed strong immunoreactivity for the α-isoform in parietal cells and for the β-isoform in pit cells of the stomach, along with the presence of these proteins in various other sites. Comparative staining revealed a similar but not identical distribution of IκB proteins in the Mongolian gerbil, a rodent model for H. pylori infection. The findings suggest that the α- and β-iso-forms are dominant IκB proteins in gastric parietal and foveolar cells, respectively, and point to a role for these transcription regulators in modulating pathological responses in stomach and other organs.
Nidogen-1: Expression and Ultrastructural Localization During the Onset of Mesoderm Formation in the Early Mouse Embryo
Authors: Miosge Nicolai , Quondamatteo Fabio , Klenczar Christina , Herken Rainer
Published: April 2011

Nidogen-1, a key component of basement membranes, is considered to function as a link between laminin and collagen Type IV networks and is expressed by mesenchymal cells during embryonic and fetal development. It is not clear which cells produce nidogen-1 in early developmental stages when no mesenchyme is present. We therefore localized nidogen-1 and its corresponding mRNA at the light and electron microscopic level in Day 7 mouse embryos during the onset of mesoderm formation by in situ hybridization, light microscopic immunostaining, and immunogold histochemistry. Nidogen-1 mRNA was found not only in the cells of the ectoderm-derived mesoderm but also in the cytoplasm of the endoderm and ectoderm, indicating that all three germ layers express it. Nidogen-1 was localized only in fully developed basement membranes of the ectoderm and was not seen in the developing endodermal basement membrane or in membranes disrupted during mesoderm formation. In contrast, laminin-1 and collagen Type IV were present in all basement membrane types at this developmental stage. The results indicate that, in the early embryo, nidogen-1 may be expressed by epithelial and mesenchymal cells, that both cell types contribute to embryonic basement membrane formation, and that nidogen-1 might serve to stabilize basement membranes in vivo.
Human Articular Chondrocytes Express Osteogenic Protein-1
Authors: Chubinskaya Susan , Merrihew Charis , Cs-Szabo Gabriella , Mollenhauer Juergen , McCartney John , Rueger David C. , Kuettner Klaus E.
Published: April 2011

This study demonstrates for the first time that human articular chondrocytes express osteogenic protein-1 (OP-1). OP-1 was originally purified from bone matrix and was shown to induce cartilage and bone formation. Both OP-1 protein and message were present in human normal and osteoarthritic (OA) cartilages. OP-1 mRNA was upregulated in OA cartilage compared with normal adult tissues. However, the level of mature OP-1 protein in the same OA tissues was downregulated, whereas the pro-OP-1 remained high. Moreover, these two forms of OP-1 were localized in an inverted manner. Mature OP-1 was primarily detected in the superficial layer, whereas the pro-form was mostly in the deep layer of cartilage. The presence of pro- and mature OP-1 in extracts of normal and OA cartilages was confirmed by Western blotting. These findings imply that articular chondrocytes continue to express and synthesize OP-1 throughout adulthood. The observed patterns of the distribution of pro- and mature OP-1 also suggest differences in the processing of this molecule by normal and OA chondrocytes and by the cells in the superficial and deep layers. Distinct distribution of OP-1 and its potential activation in deep zones and regions of cloning in OA cartilages may provide clues to the potential involvement of endogenous OP-1 in repair mechanisms.
Glial Fibrillary Acidic Protein (GFAP)-like Immunoreactivity in Rat Endocrine Pancreas
Authors: Regoli Marì , Orazioli Daniela , Gerli Renato , Bertelli Eugenio
Published: April 2011

The study of intermediate filament expression in the pancreatic epithelium has been previously focused almost exclusively on cytokeratins. Transient vimentin immunoreactivity has also been detected in duct cells of rat fetal pancreas. Here we report that, in rat pancreas, intense GFAP-like immunoreactivity is detectable in a subpopulation of endocrine cells located in the periphery of the islet of Langerhans. In addition, staining appeared to be preferentially localized to the apical pole of the cells. Two different polyclonal antibodies were employed in this study, with analogous results. Staining of consecutive sections with anti-GFAP, anti-glucagon, and anti-somatostatin antibodies demonstrates that GFAP-like immunoreactivity is present in glucagon-secreting cells. The relevance of this finding is discussed.
PCR-derived ssDNA Probes for Fluorescent In Situ Hybridization to HIV-1 RNA
Authors: Knuchel Marlyse C. , Graf Brigit , Schlaepfer Erika , Kuster Herbert , Fischer Marek , Weber Rainer , Cone Richard W.
Published: April 2011

We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 μg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH.
Immunohistochemical Localization of Taurine in the Male Reproductive Organs of the Rat
Authors: Lobo Maria V.T. , Alonso F. Javier M. , del Río Rafael Martín
Published: April 2011

The amino acid taurine has been implicated in several aspects of reproductive system physiology. However, its localization in these organs has not been previously analyzed. The aim of this study was to characterize its distribution in male rat reproductive organs by immunohistochemical methods. Taurine was localized in the smooth muscle cells of the tissues studied and in the skeletal fibers of the cremaster muscle. In the testis, taurine was found in Leydig cells, vascular endothelial cells, and other interstitial cells. No immunoreactivity was observed in the cells of the seminiferous tubules, either in germ cells at all spermatogenic stages or in Sertoli cells. However, peritubular myoid cells were immunostained. Most epithelial cells of the efferent ducts were immunolabeled, whereas the epithelial cells of the rete testis (extratesticular segments), epididymis (caput, corpus, and cauda regions), and ductus deferens were unstained. However, most epithelial cells from the intratesticular segments of the rete were immunopositive. Some cells identified as intraepithelial macrophages and lymphocytes, apical cells, and narrow cells were intensely immunolabeled. Regional differences in the distribution of these cell types along the ducts studied were also noted. The possible functional roles for taurine in these cells are discussed.
Effect of Fixation and Epitope Retrieval on BrdU Indices in Mammary Carcinomas
Authors: McGinley John N. , Knott Katrina K. , Thompson Henry J.
Published: April 2011

Studies in which 5-bromo-2′-deoxyuridine (BrdU) is used to quantify rates of cell proliferation are conducted prospectively. Therefore, the opportunity exists to select conditions that optimize detection of the BrdU epitope. The objective of this study was to quantify the extent to which the BrdU epitope was masked by formalin vs methacarn fixation in the assessment of cell proliferation. Mammary carcinomas from animals pulse-labeled with BrdU were trisected. A portion was frozen and the remaining two portions were fixed in 10% neutral buffered formalin or methacarn for 24 hr, processed, embedded in paraffin, and sections stained for incorporated BrdU using a peroxidase immunohistochemical staining technique. Antigen retrieval techniques also were applied to formalin-fixed sections. Fixation in methacarn gave the highest labeling index (16.4%), which was comparable to that observed in unfixed frozen sections (17.5%). Formalin fixation alone dramatically suppressed the labeling index (0.3%), which was only partially recovered using various antigen retrieval techniques (2.1-8.1%). Methacarn fixation is recommended for prospective studies in which BrdU detection is planned because of the quantitative recovery of epitope and the simplicity of the approach.
Integrin Expression during Epithelial Migration and Restratification in the Tenascin-C-deficient Mouse Cornea
Authors: Iglesia Drina D. Sta , Gala Purvi H. , Qiu Tianqing , Stepp Mary Ann
Published: April 2011

In the unwounded cornea, tenascin-C localizes to a short stretch of the basement membrane zone at the corneoscleral junction or limbus. To determine whether the function of the limbus is affected by the absence of tenascin-C, mice possessing a deletion of tenascin-C and strain-matched wild-type mice are used in corneal debridement wounding experiments. The expression of integrins (α3, α9, and β4) in the tenascin-C knockout corneas is evaluated by producing polyclonal cytoplasmic domain antipeptide sera and performing immunofluorescence microscopy. In addition, we evaluate the localization of several other proteins involved in wound healing, including fibronectin, laminin β1, nidogen/ entactin, and VCAM-1, in both the tenascin knockout and wild-type mice. There are no differences in healing rate, scarring, or neovascularization after corneal debridement wounds. α9 integrin is expressed at the limbal border of unwounded tenascin-C knockout animals and is upregulated during migration only after the larger wounds. At 8 weeks after larger wounds, the localization of α9 again becomes restricted to the limbal border. Results show that tenascin-C is not required for development or maintenance of the corneal limbus or for normal re-epithelialization of corneal epithelial cells after debridement wounding.
Immunohistochemical Study of the Expression of MUC6 Mucin and Co-expression of Other Secreted Mucins (MUC5AC and MUC2) in Human Gastric Carcinomas
Authors: Reis Celso A. , David Leonor , Carvalho Filipa , Mandel Ulla , Bolós Carme de , Mirgorodskaya Ekaterina , Clausen Henrik , Sobrinho-Simões Manuel
Published: April 2011

To investigate the expression of MUC6 mucin in gastric carcinomas, we generated a novel monoclonal antibody (MAb CLH5) using an MUC6 synthetic peptide. MAb CLH5 reacted exclusively with the MUC6 peptide and with native and deglycosylated mucin extracts from gastric tissues. MAb CLH5 immunoreactivity was observed in normal gastric mucosa restricted to pyloric glands of the antrum and mucopeptic cells of the neck zone of the body region. In a series of 104 gastric carcinomas, 31 (29.8%) were immunoreactive for MUC6. The expression of MUC6 was not associated with histomorphological type or with clinicopathological features of the carcinomas. Analysis of the co-expression of MUC6 with other secreted mucins (MUC5AC and MUC2) in 20 gastric carcinomas revealed that different mucin core proteins are co-expressed in 55% of the cases. MUC6 was co-expressed and co-localized with MUC5AC in 45% and with MUC2 in 5% of the cases. Expression of MUC2 alone was observed in 25% of the cases. All carcinomas expressing MUC2 mucin in more than 50% of the cells were of the mucinous type according to the WHO classification. The co-expression of mucins was independent of the histomorphological type and stage of the tumors. In conclusion, we observed, using a novel well-characterized MAb, that MUC6 is a good marker of mucopeptic cell differentiation and is expressed in 30% of gastric carcinomas, independent of the clinicopathological features of the cases. Furthermore, we found that co-expression and co-localization of mucins in gastric carcinomas is independent of histomorphology and staging. Finally, we observed that intestinal mucin MUC2 is expressed as the most prominent mucin of the mucins tested in mucinous-type gastric carcinomas.
Immunolocalization of Collagen Types II and III in Single Fibrils of Human Articular Cartilage
Authors: Young Robert D. , Lawrence Paul A. , Duance Victor C. , Aigner Thomas , Monaghan Paul
Published: April 2011

Type II and III fibrillar collagens were localized by immunogold electron microscopy in resin sections of human femoral articular cartilage taken from the upper radial zone in specimens from patients with osteoarthritis. Tissue samples stabilized by high-pressure cryofixation were processed by freeze-substitution, either in acetone containing osmium or in methanol without chemical fixatives, before embedding in epoxy or Lowicryl resin, respectively. Ultrastructural preservation was superior with osmium-acetone, although it was not possible to localize collagens by this method. In contrast, in tissue prepared by low-temperature methods without chemical fixation, collagens were successfully localized with mono- or polyclonal antibodies to the helical (Types II and III) and amino-propeptide (Type III procollagen) domains of the molecule. Dual localization using secondary antibodies labeled with 5- or 10-nm gold particles demonstrated the presence of Types II and III collagen associated within single periodic banded fibrils. Collagen fibrils in articular cartilage are understood to be heteropolymers mainly of Types II, IX, and XI collagen. Our observations provide further evidence for the complexity of these assemblies, with the potential for interactions between at least 11 distinct collagen types as well as several non-collagenous components of the extracellular matrix.
Contributions of Immunohistochemistry and In Situ Hybridization to the Functional Analysis of Pituitary Adenomas
Authors: Osamura R. Yoshiyuki , Tahara Shigeyuki , Kurotani Reiko , Sanno Naoko , Matsuno Akira , Teramoto Akira
Published: April 2011

Immunohistochemistry (IHC) and recently in situ hybridization (ISH) have elucidated various aspects of human pituitary adenomas, i.e., functional differentiation and classification, transcription factors and mechanism of hormone production, regulation of hormone secretion, and processing of prohormones. Recently, the use of tyramide (catalyzed signal amplification; TSA or CSA) and RT-PCR has been effective for detection of trivial amount of proteins (peptides) and mRNA, respectively. Immunomolecular histochemistry is expected to further clarify the function and biology of human pituitary adenomas.e
Metabolic Imaging in Multicellular Spheroids of Oncogene-transfected Fibroblasts
Authors: Walenta Stefan , Doetsch Joerg , Mueller-Klieser Wolfgang , Kunz-Schughart Leoni A.
Published: April 2011

Four rat embryo fibroblast (REF) cell lines with defined oncogenic transformation were used to study the relationship between tumorigenic conversion, metabolism, and development of cell death in a 3D spheroid system. Rat1 (spontaneously immortalized) and M1 ( myc-transfected) fibroblasts represent early nontumorigenic transformation stages, whereas Rat1-T1 (T24Ha- ras-transfected Rat1) and MR1 ( myc/T24Ha- ras-co-transfected REF) cells express a highly tumorigenic phenotype. Localized ATP, glucose, and lactate concentrations in spheroid median sections were determined by imaging bioluminescence. ATP concentrations were low in the nonproliferating Rat1 aggregates despite sufficient oxygen and glucose availability and lack of lactate accumulation. In MR1 spheroids, a 50% decrease in central ATP preceded the development of central necrosis at a spheroid diameter of around 800 μm. In contrast, the histomorphological emergence of cell death at a diameter of around 500 μm in Rat1-T1 spheroids coincided with an initial steep drop in ATP. Concomitantly, reduction in central glucose and increase in lactate before cell death were recorded in MR1 but not in Rat1-T1 spheroids. As shown earlier, myc transfection confers a considerable resistance to hypoxia of MR1 cells in the center of spheroids, which is reflected by their capability to maintain cell integrity and ATP content in a hypoxic environment. The data obtained suggest that small alterations in the genotype of tumor cell lines, such as differences in the immortalization process, lead to substantial differences in morphological structure, metabolism, occurrence of cell death, and tolerance to hypoxia in spheroid culture.
Cell Wall-associated Protein A as a Tool for Immunolocalization of Staphylococcus aureus in Infected Human Airway Epithelium
Authors: Mongodin Emmanuel , Bajolet Odile , Hinnrasky Jocelyne , Puchelle Edith , de Bentzmann Sophie
Published: April 2011

Staphylococcus aureus is a common human pathogen involved in non-bronchial diseases and in genetic and acquired bronchial diseases. In this study, we applied an immunolabeling approach for in vivo and in vitro detection of S. aureus, based on the affinity of staphylococcal protein A (SpA) for the Fc region of immunoglobulins, especially IgG. Most strains of S. aureus, including clinical strains, can be detected with this labeling technique. The approach can be used for detection and localization with transmission electron microscopy or light-fluorescence microscopy of S. aureus in infected tissues such as human bronchial tissue from cystic fibrosis (CF) patients. The methodology can also be applied to cell culture models with the aim of characterizing bacterial adherence to epithelial cells in backscattered electron imaging with scanning electron microscopy. Application to the study of S. aureus adherence to airway epithelium showed that the bacteria did not adhere in vivo to intact airway epithelium. In contrast, bacteria adhered to the basolateral plasma membrane of columnar cells, to basal cells, to the basement membrane and were identified beneath the lamina propria when the epithelium was injured and remodeled, or in vitro when the epithelial cells were dedifferentiated.
Cellular and Subcellular Localization of Catalase in the Heart of Transgenic Mice
Authors: Zhou Zhanxiang , Kang Y. James
Published: April 2011

SUMMARY Previous studies have described a cardiac-specific, catalase-overexpressing transgenic mouse model that was used to study myocardial oxidative injury. This study was undertaken to demonstrate cellular and subcellular localization of catalase in the hearts of transgenic mice. By the light microscopic immunoperoxidase method, we found that the overexpressed catalase was exclusively localized in cardiomyocytes. The ratios of immunoreactive cardiomyocytes in the heart were quite different among three transgenic lines examined but agreed with the elevated levels of catalase activity. In the cardiac blood vessels, positive cells were found in the walls of pulmonary veins and the vena cava, which consist of cardiomyocytes, but not in the pulmonary arteries, aorta, or cardiac valves. The electron microscopic immunogold method revealed that the elevated catalase was in sarcoplasm, nucleus, and peroxisomes, but not in mitochondria. In contrast to these distributions, catalase in the non-transgenic cardiomyocytes was in peroxisomes only. In addition, the number and size of peroxisomes in the transgenic cardiomyocytes were markedly increased, but no other ultrastructural changes were observed in comparison with those of non-transgenic mice. These results demonstrated that the elevated catalase in transgenic mouse heart is localized in cardiomyocytes and is distributed to peroxisomal and extraperoxisomal, but not mitochondrial, compartments.
Immunocytochemical Localization of the Extracellular Calcium-Sensing Receptor in Normal and Malignant Human Large Intestinal Mucosa
Authors: Sheinin Yuri , Kállay Enikö , Wrba Friedrich , Kriwanek Stefan , Peterlik Meinrad , Cross Heide S.
Published: April 2011

SUMMARY We identified the parathyroid type Ca2+-sensing receptor (CaR) in normal human colon mucosa and in cancerous lesions at the mRNA and protein level. Polymerase chain reaction produced an amplification product from reverse-transcribed large intestinal RNA which corresponded in size and length to a 537-bp sequence from exon 7 of the CaR gene. With a specific antiserum against its extracellular domain, the CaR could be detected by immunostaining in normal human colon mucosa in cells preferentially located at the crypt base. The CaR protein was also expressed in tumors of the large bowel in all 20 patients examined. However, the great majority of CaR-positive cells in the adenocarcinomas inspected were confined to more differentiated areas exhibiting glandular-tubular structures. Poorly or undifferentiated regions were either devoid of specific immunoreactivity or contained only isolated CaR-positive cells. In the normal mucosa and in glandular-tubular structures of cancerous lesions, the CaR was exclusively expressed in chromogranin A-positive enteroendocrine cells and in only a small fraction of PCNA-positive cells.
Seven-color Fluorescence Imaging of Tissue Samples Based on Fourier Spectroscopy and Singular Value Decomposition
Authors: Tsurui Hiromichi , Nishimura Hiroyuki , Hattori Susumu , Hirose Sachiko , Okumura Ko , Shirai Toshikazu
Published: April 2011

SUMMARY Seven-color analyses of immunofluorescence-stained tissue samples were accomplished using Fourier spectroscopy-based hyperspectral imaging and singular value decomposition. This system consists of a combination of seven fluorescent dyes, three filter-sets, an epifluorescence microscope, a spectral imaging system, a computer for data acquisition, and data analysis software. The spectra of all pixels in a multicolor image were taken simultaneously using a Sagnac type interferometer. The spectra were deconvolved to estimate the contribution of each component dye, and individual dye images were constructed based on the intensities of assigned signals. To obtain mixed spectra, three filter sets, i.e., Bl, Gr, and Rd for Alexa488 and Alexa532, for Alexa546, Alexa568, and Alexa594, and for Cy5 and Cy5.5, respectively, were used for simultaneous excitation of two or three dyes. These fluorophores have considerable spectral overlap which precludes their separation by conventional analysis. We resolved their relative contributions to the fluorescent signal by a method involving linear unmixing based on singular value decomposition of the matrices consisting of dye spectra. Analyses of mouse thymic tissues stained with seven different fluorescent dyes provided clear independent images, and any combination of two or three individual dye images could be used for constructing multicolor images.