bluuebunny/crossref_metadata_2025_split huggingface dataset:
Criteria for effective design, construction, and gene knockdown by shRNA vectors
Authors: Taxman Debra J , Livingstone Laura R , Zhang Jinghua , Conti Brian J , Iocca Heather A , Williams Kristi L , Lich John D , Ting Jenny P-Y , Reed William 
Published: January 2006

Abstract

Background
RNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized.

Results
To determine whether published algorithms for siRNA oligonucleotide design apply to shRNA, we constructed 27 shRNAs from 11 human genes expressed stably using retroviral vectors. We demonstrate an efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids. We show that sequencing through shRNA vectors can be problematic due to the intrinsic secondary structure of the hairpin, and we determine a strategy for effective sequencing by using a combination of modified BigDye chemistries and DNA relaxing agents. The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. Our results show that none of the scoring algorithms can explain a significant percentage of variance in shRNA knockdown efficacy as assessed by linear regression analysis or ROC curve analysis. Application of a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms. Analysis of an independent set of data from 38 shRNAs pooled from previous publications confirms these findings.

Conclusion
The use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors.
Virtual cooperativity in myoglobin oxygen saturation curve in skeletal muscle in vivo
Authors: Seiyama Akitoshi 
Published: January 2006

Abstract

Background
Myoglobin (Mb) is the simplest monomeric hemoprotein and its physicochemical properties including reversible oxygen (O2)binding in aqueous solution are well known. Unexpectedly, however, its physiological role in intact muscle has not yet been established in spite of the fact that the role of the more complex tetrameric hemoprotein, hemoglobin (Hb), in red cells is well established. Here, I report my new findings on an overlooked property of skeletal Mb.

Methods
I directly observed the oxygenation of Mb in perfused rat skeletal muscle under various states of tissue respiration. A computer-controlled rapid scanning spectrophotometer was used to measure the oxygenation of Mb in the transmission mode. The light beam was focused on the thigh (quadriceps) through a 5-mm-diameter light guide. The transmitted light was conducted to the spectrophotometer through another 5-mm-diameter light guide. Visible difference spectra in the range of 500–650 nm were recorded when O2 uptake in the hindlimb muscle reached a constant value after every stepwise change in the O2 concentration of the buffer.

Results
The O2 dissociation curve (ODC) of Mb, when the effluent buffer O2 pressure was used as the abscissa, was of a sigmoid shape under normal and increased respiratory conditions whereas it was of rectangular hyperbolic shape under a suppressed respiratory condition. The dissociation curve was shifted toward the right and became more sigmoid with an increase in tissue respiration activity. These observations indicate that an increase in O2 demand in tissues makes the O2 saturation of Mb more sensitive to O2 pressure change in the capillaries and enhances the Mb-mediated O2 transfer from Hb to cytochrome oxidase (Cyt. aa3), especially under heavy O2 demands.

Conclusion
The virtual cooperativity and O2 demand-dependent shifts of the ODC may provide a basis for explaining why Mb has been preserved as monomer during molecular evolution.
Recruitment of aged donor heart with pharmacological stress echo. A case report
Authors: Arpesella Giorgio , Gherardi Sonia , Bombardini Tonino , Picano Eugenio 
Published: January 2006

Abstract

Background
The heart transplant is a treatment of the heart failure, which is not responding to medications, and its efficiency is already proved: unfortunately, organ donation is a limiting step of this life-saving procedure.
To counteract heart donor shortage, we should screen aged potential donor hearts for initial cardiomyopathy and functionally significant coronary artery disease.
Donors with a history of cardiac disease are generally excluded. Coronary angiography is recommended for most male donors older than 45 years and female donors older than 50 years to evaluate coronary artery stenoses. A simpler way to screen aged potential donor hearts for initial cardiomyopathy and functionally significant coronary artery disease should be stress echocardiography.

Case report
A marginal donor (A 57 year old woman meeting legal requirements for brain death) underwent a transesophageal (TE) Dipyridamole stress echo (6 minutes accelerated protocol) to rule out moderate or severe heart and coronary artery disease. Wall motion was normal at baseline and at peak stress (WMSI = 1 at baseline and peak stress, without signs of stress inducible ischemia). The pressure/volume ratio was 9.6 mmHg/ml/m2 at baseline, increasing to 14 mmHg/ml/m2 at peak stress, demonstrating absence of latent myocardial dysfunction.
The marginal donor heart was transplanted to a recipient "marginal" for co-morbidity ( a 63 year old man with multiple myeloma and cardiac amyloidosis , chronic severe heart failure, NYHA class IV).
Postoperative treatment and early immunosuppressant regimen were performed according to standard protocols.
The transplanted heart was assessed normal for dimensions and ventricular function at transthoracic (TT) echocardiography on post-transplant day 7.
Coronary artery disease was ruled out at coronary angiography one month after transplant; left ventriculography showed normal global and segmental LV function of the transplanted heart.

Conclusion
For the first time stress echo was successfully used in the critical theater of screening potential donor hearts. This method is enormously more feasible, less expensive, and more environmentally sustainable than any possible alternative strategy based on stress scintigraphy perfusion imaging or coronary angiography. The selection of hearts "too good to die" on the basis of bedside resting and stress echo can be a critical way to solve the mismatch between donor need and supply.
Patterns of drug use among a sample of drug users and injecting drug users attending a General Practice in Iran
Authors: Day Carolyn , Nassirimanesh Bijan , Shakeshaft Anthony , Dolan Kate 
Published: January 2006

Abstract

Aim
This study aimed to examine drug use, drug treatment history and risk behaviour among a sample of Iranian drug users seeking treatment through a general practice clinic in Iran.

Methods
Review of medical records and an intake questionnaire at a large general practice in Marvdasht, Iran, with a special interest in drug dependence treatment. Records from a random sample of injecting drug users (IDU), non-injecting drug users (DU) and non-drug using patients were examined.

Results
292 records were reviewed (34% IDU, 31% DU and 35% non-drug users). Eighty-three percent were males; all females were non-drug users. The mean age of the sample was 30 years. Of the IDU sample, 67% reported sharing a needle or syringe, 19% of these had done so in prison. Of those who had ever used drugs, being 'tired' of drug use was the most common reason for seeking help (34%). Mean age of first drug use was 20 years. The first drugs most commonly used were opium (72%), heroin (13%) and hashish/ other cannabinoids (13%). Three quarters reported having previously attempted to cease their drug use. IDU were more likely than DU to report having ever been imprisoned (41% vs 7%) and 41% to have used drugs in prison.

Conclusion
This study has shown that there is a need for general practice clinics in Iran to treat drug users including those who inject and that a substantial proportion of those who inject have shared needles and syringes, placing them at risk of BBVI such as HIV and hepatitis C. The expansion of services for drug users in Iran such as needle and syringe programs and pharmacotherapies are likely to be effective in reducing the harms associated with opium use and heroin injection.
Gender differences in health-related quality of life of adolescents with cystic fibrosis
Authors: Arrington-Sanders Renata , Yi Michael S , Tsevat Joel , Wilmott Robert W , Mrus Joseph M , Britto Maria T 
Published: January 2006

AbstractBackgroundFemale patients with cystic fibrosis (CF) have consistently poorer survival rates than males across all ages. To determine if gender differences exist in health-related quality of life (HRQOL) of adolescent patients with CF, we performed a cross-section analysis of CF patients recruited from 2 medical centers in 2 cities during 1997–2001.MethodsWe used the 87-item child self-report form of the Child Health Questionnaire to measure 12 health domains. Data was also collected on age and forced expiratory volume in 1 second (FEV1). We analyzed data from 98 subjects and performed univariate analyses and linear regression or ordinal logistic regression for multivariable analyses.ResultsThe mean (SD) age was 14.6 (2.5) years; 50 (51.0%) were female; and mean FEV1was 71.6% (25.6%) of predicted. There were no statistically significant gender differences in age or FEV1. In univariate analyses, females reported significantly poorer HRQOL in 5 of the 12 domains. In multivariable analyses controlling for FEV1and age, we found that female gender was associated with significantly lower global health (p < 0.05), mental health (p < 0.01), and general health perceptions (p < 0.05) scores.ConclusionFurther research will need to focus on the causes of these differences in HRQOL and on potential interventions to improve HRQOL of adolescent patients with CF.
Disaggregation and invasion of ovarian carcinoma ascites spheroids
Authors: Burleson Kathryn M , Boente Matthew P , Pambuccian Stefan E , Skubitz Amy PN 
Published: January 2006

Abstract

Background
Malignant ascites often develops in advanced stages of ovarian carcinoma, consisting of single and aggregated tumor cells, or spheroids. Spheroids have commonly been used as tumor models to study drug efficacy, and have shown resistance to some chemotherapies and radiation. However, little is known about the adhesive or invasive capabilities of spheroids, and whether this particular cellular component of the ascites can contribute to dissemination of ovarian cancer. Here, we examined the invasive ability of ascites spheroids recovered from seven ovarian carcinoma patients and one primary peritoneal carcinoma (PPC) patient.

Methods
Ascites spheroids were isolated from patients, purified, and immunohistochemical analyses were performed by a pathologist to confirm diagnosis. In vitro assays were designed to quantify spheroid disaggregation on a variety of extracellular matrices and dissemination on and invasion into normal human mesothelial cell monolayers. Cell proliferation and viability were determined in each assay, and statistical significance demonstrated by the student's t-test.

Results
Spheroids from all of the patients' ascites samples disaggregated on extracellular matrix components, with the PPC spheroids capable of complete disaggregation on type I collagen. Additionally, all of the ascites spheroid samples adhered to and disaggregated on live human mesothelial cell monolayers, typically without invading them. However, the PPC ascites spheroids and one ovarian carcinoma ascites spheroid sample occasionally formed invasive foci in the mesothelial cell monolayers, suggestive of a more invasive phenotype.

Conclusion
We present here in vitro assays using ascites spheroids that imitate the spread of ovarian cancer in vivo. Our results suggest that systematic studies of the ascites cellular content are necessary to understand the biology of ovarian carcinoma.
Effect of lateral perturbations on psychophysical acceleration detection thresholds
Authors: Richerson Samantha J , Morstatt Scott M , O'Neal Kristopher K , Patrick Gloria , Robinson Charles J 
Published: January 2006

Abstract

Background
In understanding how the human body perceives and responds to small slip-like motions, information on how one senses the slip is essential. The effect of aging and plantar sensory loss on detection of a slip can also be studied. Using psychophysical procedures, acceleration detection thresholds of small lateral whole-body perturbations were measured for healthy young adults (HYA), healthy older adults (HOA) and older adults with diabetic neuropathy (DOA). It was hypothesized that young adults would require smaller accelerations than HOA's and DOA's to detect perturbations at a given displacement.

Methods
Acceleration detection thresholds to whole-body lateral perturbations of 1, 2, 4, 8, and 16 mm were measured for HYAs, HOAs, and DOAs using psychophysical procedures including a two-alternative forced choice protocol. Based on the subject's detection of the previous trial, the acceleration magnitude of the subsequent trial was increased or decreased according to the parameter estimation by sequential testing methodology. This stair-stepping procedure allowed acceleration thresholds to be measured for each displacement.

Results
Results indicate that for lateral displacements of 1 and 2 mm, HOAs and DOAs have significantly higher acceleration detection thresholds than young adults. At displacements of 8 and 16 mm, no differences in threshold were found among groups or between the two perturbation distances. The relationship between the acceleration threshold and perturbation displacement is of particular interest. Peak acceleration thresholds of approximately 10 mm/s2 were found at displacements of 2, 4, 8, and 16 mm for HYAs; at displacements of 4, 8, and 16 mm for HOAs; and at displacements of 8 and 16 mm for DOAs. Thus, 2, 4, and 8 mm appear to be critical breakpoints for HYAs, HOAs, and DOAs respectively, where the psychometric curve deviated from a negative power law relationship. These critical breakpoints likely indicate a change in the physiology of the system as it responds to the stimuli.

Conclusion
As a function of age, the displacement at which the group deviates from a negative power law relationship increases from 2 mm to 4 mm. Additionally, the displacement at which subjects with peripheral sensory deficits deviate from the negative power law relations increases to 8 mm. These increases as a function of age and peripheral sensory loss may help explain the mechanism of falls in the elderly and diabetic populations.
Hydrocortisone and Purinergic Signaling Stimulate Sodium/Iodide Symporter (NIS)-Mediated Iodide Transport in Breast Cancer Cells
Authors: Dohán Orsolya , De la Vieja Antonio , Carrasco Nancy 
Published: January 2006

AbstractThe sodium/iodide symporter (NIS) mediates a remarkably effective targeted radioiodide therapy in thyroid cancer; this approach is an emerging candidate for treating other cancers that express NIS, whether endogenously or by exogenous gene transfer. Thus far, the only extrathyroidal malignancy known to express functional NIS endogenously is breast cancer. Therapeutic efficacy in thyroid cancer requires that radioiodide uptake be maximized in tumor cells by manipulating well-known regulatory factors of NIS expression in thyroid cells, such as TSH, which stimulates NIS expression via cAMP. Similarly, therapeutic efficacy in breast cancer will likely depend on manipulating NIS regulation in mammary cells, which differs from that in the thyroid. Human breast adenocarcinoma MCF-7 cells modestly express endogenous NIS when treated with all-trans-retinoic acid (tRa). We report here that hydrocortisone and ATP each markedly stimulates tRa-induced NIS protein expression and plasma membrane targeting in MCF-7 cells, leading to at least a 100% increase in iodide uptake. Surprisingly, the adenyl cyclase activator forskolin, which promotes NIS expression in thyroid cells, markedly decreases tRa-induced NIS protein expression in MCF-7 cells. Isobutylmethylxanthine increases tRa-induced NIS expression in MCF-7 cells, probably through a purinergic signaling system independent of isobutylmethylxanthine’s action as a phosphodiesterase inhibitor. We also observed that neither iodide, which at high concentrations down-regulates NIS in the thyroid, nor cAMP has a significant effect on NIS expression in MCF-7 cells. Our findings may open new strategies for breast-selective pharmacological modulation of functional NIS expression, thus improving the feasibility of using radioiodide to effectively treat breast cancer.
Detergent-resistant membrane domains but not the proteasome are involved in the misfolding of a PrP mutant retained in the endoplasmic reticulum
Authors: Campana Vincenza , Sarnataro Daniela , Fasano Carlo , Casanova Philippe , Paladino Simona , Zurzolo Chiara 
Published: January 2006

Inherited prion diseases are neurodegenerative pathologies related to genetic mutations in the prion protein (PrP) gene, which favour the conversion of PrPC into a conformationally altered pathogenic form, PrPSc. The molecular basis of PrPC/PrPSc conversion, the intracellular compartment where it occurs and how this process leads to neurological dysfunction are not yet known.
We have studied the intracellular synthesis, degradation and localization of a PrP mutant associated with a genetic form of Creutzfeldt-Jakob disease (CJD), PrPT182A, in transfected FRT cells. PrPT182A is retained in the endoplasmic reticulum (ER), is mainly associated with detergent-resistant microdomains (DRMs) and is partially resistant to proteinase K digestion. Although an untranslocated form of this mutant is polyubiquitylated and undergoes ER-associated degradation, the proteasome is not responsible for the degradation of its misfolded form, suggesting that it does not have a role in the pathogenesis of inherited diseases. On the contrary, impairment of PrPT182A association with DRMs by cholesterol depletion leads to its accumulation in the ER and substantially increases its misfolding. These data support the previous hypothesis that DRMs are important for the correct folding of PrP and suggest that they might have a protective role in pathological scrapie-like conversion of PrP mutants.
The β2-adrenergic receptor activates pro-migratory and pro-proliferative pathways in dermal fibroblasts via divergent mechanisms
Authors: Pullar Christine E. , Isseroff R. Rivkah 
Published: January 2006

Dermal fibroblasts are required for skin wound repair; they migrate into the wound bed, proliferate, synthesize extracellular matrix components and contract the wound. Although fibroblasts express β2-adrenergic receptors (β2-AR) and cutaneous keratinocytes can synthesize β-AR agonists (catecholamines), the functional significance of this hormonal mediator network in the skin has not been addressed. Emerging studies from our laboratory demonstrate that β2-AR activation modulates keratinocyte migration, essential for wound re-epithelialization. Here we describe an investigation of the effects of β2-AR activation on the dermal component of wound healing. We examined β2-AR-mediated regulation of biological processes in dermal fibroblasts that are critical for wound repair: migration, proliferation, contractile ability and cytoskeletal conformation.We provide evidence for the activation of at least two divergent β2-AR-mediated signaling pathways in dermal fibroblasts, a Src-dependent pro-migratory pathway, transduced through the epidermal growth factor receptor and extracellular signal-regulated kinase, and a PKA-dependent pro-proliferative pathway. β2-AR activation attenuates collagen gel contraction and alters the actin cytoskeleton and focal adhesion distribution through PKA-dependent mechanisms. Our work uncovers a previously unrecognized role for the adrenergic hormonal mediator network in the cutaneous wound repair process. Exploiting these divergent β2-AR agonist responses in cutaneous cells may generate novel therapeutic approaches for the control of wound healing.
Newborn Screening for Hepatorenal Tyrosinemia: Tandem Mass Spectrometric Quantification of Succinylacetone
Authors: Sander Johannes , Janzen Nils , Peter Michael , Sander Stefanie , Steuerwald Ulrike , Holtkamp Ute , Schwahn Bernd , Mayatepek Ertan , Trefz Friedrich K , Das Anibh M 
Published: January 2006

AbstractBackground: False-positive and false-negative results occur in current newborn-screening programs for hepatorenal tyrosinemia, which measure tyrosine concentrations in blood spots, sometimes in combination with other metabolites, including succinylacetone. We present our experience with a newly described method for succinylacetone quantification in routine newborn screening.Methods: Succinylacetone was extracted from blood spots that had already been extracted with absolute methanol for acylcarnitine and amino acid analysis. The solvent was acetonitrile–water (80:20 by volume) containing formic acid, hydrazine hydrate, and 100 nmol/L 5,7-dioxooctanoic acid as internal standard. Analysis was performed by tandem mass spectrometry in a separate run.Results: Of 61 344 samples, 99.6% had succinylacetone concentrations ≤5 μmol/L. With a cutoff of 10 μmol/L, no false-positive results were obtained. In 2 patients, the succinylacetone concentrations in the dried blood spots from the 36th and 56th hours of life were 152 and 271 μmol/L, respectively, and the tyrosine concentrations were 54 and 129 μmol/L. Hepatorenal tyrosinemia was subsequently confirmed in both patients. Retrospective analysis of the neonatal screening samples of 2 additional known patients revealed increased succinylacetone concentrations of 46 and 169 μmol/L, respectively.Conclusions: Tandem mass spectrometric quantification directly from residual blood spots is a useful method for the early detection of hepatorenal tyrosinemia in newborn-screening programs.