gebhart/patrepeval huggingface dataset:
Lentiviral-mediated gene transfer into human lymphocytes: role of HIV-1 accessory proteins (2000)

Resting lymphocytes are refractory to gene transfer using Moloney murine leukemia virus (MMLV)-based retroviral vectors because of their quiescent status. Recently, it has been shown that lentiviral vectors are capable of transferring genes into nondividing and terminally differentiated cells. We used human immunodeficiency virus type-1 (HIV-1)-based vectors expressing enhanced green fluorescent protein (EGFP) driven by different promoters (CMV, MPSV, or PGK) and investigated their ability to transduce human T- and B-cell lines, as well as resting or activated primary peripheral and umbilical cord blood lymphocytes. The effects of the presence or the absence of HIV-1 accessory proteins (Vif, Vpr, Vpu, and Nef) in the vector system were also assessed. Flow cytometry analysis showed no differences in the ability of these vectors of transferring the reporter gene into lymphocytic lines and mitogen-stimulated primary lymphocytes in the presence or the absence of HIV-1 accessory proteins (APs). Similarly, viral supernatants generated in the presence of accessory genes could efficiently transduce various subsets of resting lymphocytes and provide long-term expression of the transgene. No significant transduction-induced changes in cell activation or cycling status were observed and Alu-HIV-1 long terminal repeat polymerase chain reaction (LTR PCR) analysis demonstrated integration of the vector sequences at the molecular level. In contrast, in the absence of HIV-1 APs, lentiviral vectors failed to integrate and express the transgene in resting lymphocytes. These results show that transduction of primary resting lymphocytes with HIV-1-based vectors requires the presence of viral accessory proteins. (Blood. 2000;96:1309-1316)
Human Embryonic Stem Cells Are Prone to Generate Primitive, Undifferentiated Tumors in Engrafted Human Fetal Tissues in Severe Combined Immunodeficient Mice (2007)

Embryonic stem (ES) cells are uniquely endowed with the capacity of self-renewal and the potential to give rise to all possible cell types. Their differentiation potential has raised hope that these cells could be used as a renewable source for cell transplantation in severe degenerative diseases. However, progress in this direction is still limited. Using two human embryonic stem (ES) cell lines, H1 and HSF-6, and three types of human fetal tissues--thymus, lung and pancreas-we investigated whether engrafted human fetal tissues in severe combined immunodeficient mice (SCID) mice could provide a physiologically-relevant microenvironment for human ES cells to differentiate into mature cells of corresponding tissues. Surprisingly, we observed an aggressive growth of tumors when human ES cells were injected into engrafted human fetal tissues in SCID mice. These tumors displayed histological characteristics of primitive, undifferentiated tumors rather than differentiated teratomas. Additionally, these tumors exhibited a normal karyotype and did not express the characteristic antigens of embryonic carcinomas. We also found differences among human fetal tissue types in their abilities to support the growth of these primitive tumors. Our study supports and validates a previously reported phenomenon in mouse that tumorigenesis of ES cells is host dependent. Our study is also the first report to demonstrate that human ES cells are prone to generate primitive, undifferentiated tumors in human fetal tissue grafts in SCID mice and raises a potential safety concern for using human ES cell-derived cell products in humans.
Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion sperm (1988)

Transmission electron microscopy was used to confirm that a monoclonal antibody (F79.3E2; class IgG1 kappa) was specifically localized to an antigen in the acrosomal ground substance of stallion sperm. This antibody was used to develop and validate an indirect immunofluorescent procedure to evaluate integrity of the plasma-acrosomal membranes of stallion sperm. The concept was that primary monoclonal antibody would be "shielded" from its acrosomal antigen by an intact plasma membrane. Conversely, sperm with damaged plasma-acrosomal membranes would exhibit green acrosomal fluorescence when viewed with an epifluorescence microscope. A lipophilic counterstain (red fluorescence) was used to insure that all sperm were visualized. Sperm in fresh-extended or frozen-thawed semen were incubated with hybridoma supernatant containing monoclonal antibody for 30 min at 37 degrees C, then a second antibody (rabbit anti-mouse IgG-FITC) was added for 30 min at 37 degrees C. Unbound antibody was removed by dilution and centrifugation. Sperm were resuspended in phosphate-buffered saline containing Evan's blue as a counterstain. All sperm fluoresced bright red, regardless of the status of cell membranes, except that in cells with damaged plasma-acrosomal membranes, the green fluorescence associated with antibody was overriding for the rostral portion. By counting fluorescent and nonfluorescent "acrosomes", the percentage of sperm with intact plasma-acrosomal membranes was easily determined. Evaluation of five mixtures of undamaged and damaged sperm by this procedure gave a correlation of 0.91 between the percentage of damaged sperm in a mixture and the percentage of sperm with a fluorescent acrosome. Intra- and interassay coefficients of variability were less than 6%.
LAT1 gene variants—potential factors influencing the clinical course of phenylketonuria (2006)

The LAT1 amino acid exchanger is responsible for phenylalanine transport across the blood-brain barrier. As phenylalanine excess in the brain leads to mental retardation in untreated patients with phenylketonuria, mutations of the LAT1 gene can be responsible for milder clinical manifestations (lower brain toxicity of hyperphenylalaninaemia) observed in some patients. The aim of this study was to screen for LAT1 gene mutations in phenylketonuric patients. Within the coding sequence of the gene, three polymorphism and one new mutation (G41D) were detected. The protein sequence change due to the mutation described could influence the clinical course of phenylketonuria.
Mass Spectrometry, Review of the Basics: Electrospray, MALDI, and Commonly Used Mass Analyzers (2009)

Abstract Mass spectrometry (MS) has progressed to become a powerful analytical tool for both quantitative and qualitative applications. The first mass spectrometer was constructed in 1912 and since then it has developed from only analyzing small inorganic molecules to biological macromolecules, practically with no mass limitations. Proteomics research, in particular, increasingly depends on MS technologies. The ability of mass spectrometry analyzing proteins and other biological extracts is due to the advances gained through the development of soft ionization techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) that can transform biomolecules into ions. ESI can efficiently be interfaced with separation techniques enhancing its role in the life and health sciences. MALDI, however, has the advantage of producing singly charges ions of peptides and proteins, minimizing spectral complexity. Regardless of the ionization source, the sensitivity of a mass spectrometer is related to the mass analyzer where ion separation occurs. Both quadrupole and time of flight (ToF) mass analyzers are commonly used and they can be configured together as QToF tandem mass spectrometric instruments. Tandem mass spectrometry (MS/MS), as the name indicates, is the result of performing two or more sequential separations of ions usually coupling two or more mass analyzers. Coupling a quadrupole and time of flight resulted in the production of high-resolution mass spectrometers (i.e., Q-ToF). This article will historically introduce mass spectrometry and summarizes the advantages and disadvantages of ESI and MALDI along with quadrupole and ToF mass analyzers, including the technical marriage between the two analyzers. This article is educational in nature and intended for graduate students and senior biochemistry students as well as chemists and biochemists who are not familiar with mass spectrometry and would like to learn the basics; it is not intended for mass spectrometry experts. Keywords: Electrospray ionizationMALDIquadrupoletime of flightMS/MSQ-TofView correction statement:Erratum
Protein stamping for MALDI mass spectrometry using an electrowetting-based microfluidic platform (2004)

MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry) is one of the most commonly used techniques for protein analysis. In conventional systems sample preparation is typically done in well-plates and transferred onto a MALDI target by robotic systems, which are complex, huge, expensive and slow. In this paper, we present a droplet-based microfluidic interface to transfer protein samples from a well-plate format onto a MALDI target for MS analysis. The droplets are actuated using the electrowetting phenomenon, and are immersed in silicone oil which prevents non-specific adsorption and enables the manipulation of high concentrations of proteins. Droplet transport and droplet formation were evaluated as a function of protein concentration using bovine serum albumin (BSA) as a test system. Droplet transport was possible for BSA concentrations up to 10mg/mL which is three orders of magnitude higher than previously reported results on handling proteins by electrowetting. Droplet formation from on-chip reservoirs, using only electrowetting forces and no external pressure assistance, was possible up to concentrations of 0.01mg/mL. An interface between a well-plate format and the electrowetting chip, and a scheme to passively stamp droplets onto a target substrate was then designed and tested by stamping BSA solutions. In two separate experiments 3.6fmoles and 16fmoles of BSA were stamped onto a glass slide using 0.001mg/mL and 0.01mg/mL samples respectively. A protein mixture with known constituents (ABI 4700 proteomics analyzer calibration solution) was stamped onto a MALDI plate and the individual proteins were correctly identified in the mass spectrum obtained using MALDI-TOF MS. The preliminary results establish the feasibility of using an electrowetting-based microfluidic system to handle proteins especially for protein stamping applications. The proposed system has a small footprint, is easy to control, and is very fast compared to conventional robotic systems. In addition, there are no moving parts and the associated mechanical reliability issues. Future work involves scaling to a larger number of samples and integration of sample preparation steps on-chip.
A 2 $\mu\hbox{W}$ 100 nV/rtHz Chopper-Stabilized Instrumentation Amplifier for Chronic Measurement of Neural Field Potentials (2007)

This paper describes a prototype micropower instrumentation amplifier intended for chronic sensing of neural field potentials (NFPs). NFPs represent the ensemble activity of thousands of neurons and code-useful information for both normal activity and disease states. NFPs are small - of the order of tens of muV- and reside at low bandwidths that make them susceptible to excess noise. Therefore, to ensure the highest fidelity of signal measurement for diagnostic analysis, the amplifier is chopper-stabilized to eliminate 1/f and popcorn noise. The circuit was prototyped in an 0.8 mum CMOS process and consumes under 2.0 muW from a 1.8 V supply. A noise floor of 0.98 muVrms was achieved over a bandwidth from 0.05 to 100 Hz; the noise-efficiency factor of 4.6 is one of the lowest published to date. A flexible on-chip high-pass filter is used to suppress front-end electrode offsets while maintaining relevant physiological data. The monolithic architect and micropower low-noise low-supply operation could help enable applications ranging from neuroprosthetics to seizure monitors that require a small form factor and battery operation. Although the focus of this paper is on neurophysiological sensing, the circuit architecture can be applied generally to micropower sensor interfaces that benefit from chopper stabilization.
Timely Initiation of Dialysis — Single-Exchange Experience in 39 Patients Starting Peritoneal Dialysis (2002)

This pilot study has shown that a timely start of dialysis with a single overnight PD exchange is acceptable to patients. Incremental dialysis as residual renal function falls is easily managed and patients also find this acceptable. Complication and hospitalization rates were low. The presence of residual renal function often allows complications to be managed without the need for hemodialysis. The use of icodextrin as a single-exchange dialysate is associated with sterile peritonitis in a significant proportion of cases.
Mapping the SF2/ASF Binding Sites in the Bovine Growth Hormone Exonic Splicing Enhancer (2000)

Splicing of the last intron (intron D) of the bovine growth hormone pre-mRNA requires the presence of a downstream exonic splicing enhancer (ESE). This enhancer is contained within a 115-nucleotide FspI-PvuII (FP) fragment located in the middle of the last exon (exon 5). Previous work showed that the splicing factor SF2/ASF binds to this FP region and stimulates splicing of intron D in vitro. However, the precise sequences recognized by SF2/ASF within the FP region had not been determined. Here we used multiple strategies to map the SF2/ASF binding sites and determine their importance for ESE function. Taking advantage of the fact that SF2/ASF ultraviolet (UV) cross-links specifically to RNA containing the FP sequence, we first mapped a major SF2/ASF binding site by UV cross-linking and reverse transcription. This strategy identified a 29-nucleotide SF2/ASF binding region in the middle of the FP sequence containing the 7-nucleotide purine-rich motif described previously. Interestingly, this binding region is neither sufficient, nor absolutely required for SF2/ASF-mediated splicing, suggesting that additional SF2/ASF binding sites are present. The location of these additional sites was determined by electrophoretic mobility shift analysis of various subfragments of the FP sequence. Antisense 2'-O-methyl oligoribonucleotides complementary to selected SF2/ASF binding sites block bovine growth hormone intron D splicing. Thus, multiple SF2/ASF binding sites within the exonic splicing enhancer contribute to maximal enhancer activity.
The First Immunoglobulin-like Neural Cell Adhesion Molecule (NCAM) Domain Is Involved in Double-reciprocal Interaction with the Second Immunoglobulin-like NCAM Domain and in Heparin Binding (1997)

To study the function of the first immunoglobulin (Ig)-like domain of the neural cell adhesion molecule (NCAM), it was produced as a recombinant fusion protein in a bacterial expression system and as a recombinant protein in a eukaryotic expression system of the yeast Pichia pastoris. For comparison, other NCAM domains were also produced as fusion proteins. By means of surface plasmon resonance analysis, it was shown that the first Ig-like NCAM domain binds the second Ig-like NCAM domain with a dissociation constant 5.5 +/- 1.6 x 10(-5) M. Furthermore, it was found that the first Ig-like domain binds heparin. It was also demonstrated that the second Ig-like NCAM domain binds heparin and that both domains bind collagen type I via heparin but not collagen type I directly.
Expression of human phenylalanine hydroxylase activity in T lymphocytes of classical phenylketonuria children by retroviral‐mediated gene transfer (1997)

Classical phenylketonuria (PKU) is a metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene. At present, T lymphocyte-directed gene therapy is the only means for which a safety record has been established. Thus, we investigated the applicability of this strategy to PKU gene therapy. We first looked for tetrahydrobiopterin (BH4) and dihydropteridine reductase (DHPR) activity, which are required for the phenylalanine hydroxylation reaction and BH4 regeneration, respectively, in T cells isolated from PKU children. We found that T cells contained a small amount of biopterin, but significant DHPR activity, and that the intracellular biopterin content could be increased by exogenous BH4 supplementation. Moreover, PKU T cells were capable of taking up phenylalanine efficiently and effluxing acquired tyrosine. Finally, a recombinant retrovirus containing the human PAH cDNA was constructed and used to transduce isolated PKU T cells. Viral-transduced T cells produced high levels of PAH activity as compared to control mock-infected T cells. These results indicate that T lymphocytes express all that is required for synthesizing/replenishing constituents of the phenylalanine hydroxylation reaction and expressing transduced phenylalanine hydroxylase cDNA.
In vivo nanotoxicity testing using the zebrafish embryo assay (2013)

Nanoparticles are increasingly used for biomedical purposes. Many different diagnostic and therapeutic applications are envisioned for nanoparticles, but there are often also serious concerns regarding their safety. Given the fact that numerous new nanomaterials are being developed every day, and that not much is known about the long-term toxicological impact of exposure to nanoparticles, there is an urgent need to establish efficient methods for nanotoxicity testing. The zebrafish (Danio rerio) embryo assay has recently emerged as an interesting 'intermediate' method for <i>in vivo</i> nanotoxicity screening, enabling (semi-) high-throughput analyses in a system significantly more complex than cultured cells, but at the same time also less 'invasive' and less expensive than large-scale biocompatibility studies in mice or rats. The zebrafish embryo assay is relatively well-established in the environmental sciences, but it has not yet gained wide notice in the nanomedicine field. Using prototypic polymeric drug carriers, gold-based nanodiagnostics and nanotherapeutics, and iron oxide-based nanodiagnostics, we here show that toxicity testing using zebrafish embryos is easy, efficient and informative, and faithfully reflects, yet significantly extends, cell-based toxicity testing. We therefore expect that the zebrafish embryo assay will become a popular future tool for <i>in vivo</i> nanotoxicity screening.
The Nutrition Society in the 1980s: The Questionnaire Analysis (1986)

An abstract is not available for this content so a preview has been provided. As you have access to this content, a full PDF is available via the ‘Save PDF’ action button.
Contemporary Methodology for Protein Structure Determination (1984)

The techniques used for the characterization of protein and peptide structure have undergone great changes that have improved the speed, reliability, and applicability of the process. High-performance liquid chromatography and gel electrophoresis have made the purification of proteins and peptides a routine procedure, even when the compound of interest is a minor component of a complex biological mixture. The chemistry and instrumentation used in amino acid analysis and amino acid sequencing now permit the analysis of as little as 5 to 50 picomoles of samples. This represents an increase in sensitivity of more than a thousandfold over the last 10 years and has made possible the structural analysis of a wide variety of scarce but important compounds.
Pretreatment of Lignocellulosic Wastes to Improve Ethanol and Biogas Production: A Review (2008)

Lignocelluloses are often a major or sometimes the sole components of different waste streams from various industries, forestry, agriculture and municipalities. Hydrolysis of these materials is the first step for either digestion to biogas (methane) or fermentation to ethanol. However, enzymatic hydrolysis of lignocelluloses with no pretreatment is usually not so effective because of high stability of the materials to enzymatic or bacterial attacks. The present work is dedicated to reviewing the methods that have been studied for pretreatment of lignocellulosic wastes for conversion to ethanol or biogas. Effective parameters in pretreatment of lignocelluloses, such as crystallinity, accessible surface area, and protection by lignin and hemicellulose are described first. Then, several pretreatment methods are discussed and their effects on improvement in ethanol and/or biogas production are described. They include milling, irradiation, microwave, steam explosion, ammonia fiber explosion (AFEX), supercritical CO(2) and its explosion, alkaline hydrolysis, liquid hot-water pretreatment, organosolv processes, wet oxidation, ozonolysis, dilute-and concentrated-acid hydrolyses, and biological pretreatments.
Adenomatous Polyposis Coli Gene, ??-Catenin, and E-Cadherin Expression in Proximal and Distal Gastric Cancers and Precursor Lesions (2003)

The aims of this study were (1) to compare protein expression of adenomatous polyposis coli (APC) gene, beta-catenin, and E-cadherin between proximal and distal gastric adenocarcinomas and (2) to investigate their use as markers of cancer risk in intestinal metaplasia (IM). The epidemiology of proximal (cardia and gastroesophageal junction) and distal (antrum and corpus) gastric carcinomas is strikingly different despite similar morphologies. Carcinoma of the distal stomach is decreasing in incidence, whereas proximal carcinomas are increasing in incidence more than any other cancer in the Western world. This phenomenon has so far not been satisfactorily explained. IM is a well-established precursor for adenocarcinoma in the distal stomach but less so in the proximal stomach. However, its specificity as a predictor of gastric carcinoma is very low. Abnormalities of APC, beta-catenin, and E-cadherin are implicated in carcinogenesis of the stomach and may show aberrant expression at early stages of the neoplastic process. This study evaluated their immunoprofiles in 3 groups: biopsies showing normal mucosa (n = 108), biopsies showing IM (n = 99), and gastric cancer resections (n = 117). In the last group, carcinoma and noninvolved mucosa were studied. All groups included material from both proximal and distal locations. The results of this study showed that there were no differences between proximal and distal locations with regard to APC, beta-catenin, or E-cadherin expression. In both locations, high normal expression rates for all 3 molecules were present in biopsies showing normal gastric mucosa or IM and noninvolved mucosa from gastric cancer resections. In carcinomas, there was a significant decrease in both APC and E-cadherin expression, whereas beta-catenin showed abnormal cytoplasmic and nuclear staining. Diffuse-type cancers showed significantly lower E-cadherin expression than intestinal types. Noninvolved mucosa from cancer resections showed normal APC, beta-catenin, and E-cadherin expression regardless of adjacent tumor type and whether the mucosa was morphologically normal or showed IM. In conclusion, proximal and distal gastric carcinomas show no differences in expression of APC, beta-catenin, or E-cadherin; thus, the observed abnormalities do not seem to contribute to the observed epidemiologic differences between these tumors. Because loss of APC, decreased E-cadherin, or abnormal beta-catenin expression did not occur in IM, even when associated with carcinoma these immunostains are unlikely to be of value in the assessment of malignant potential in IM.