gebhart/patrepeval huggingface dataset:
The "megaprimer" method of site-directed mutagenesis. (1990)

We describe a simple and efficient method of mutagenesis which we term the "megaprimer" method. The method utilizes three oligonucleotide primers to perform two rounds of polymerase chain reaction. In the method, the product of the first polymerase chain reaction is used as one of the polymerase chain reaction primers (a "megaprimer") for the second polymerase chain reaction. When a phage promoter and a translational initiation signal are attached to the appropriate oligonucleotide primer, the mutant protein can be generated without any in vivo manipulations. To illustrate the method, two mutations in the catalytic domain of the human factor IX gene have been generated. The substitution of megaprimers for oligonucleotide primers may have utility in other polymerase chain reaction-based methods.
Biochemical Analysis of Thermotoga maritima GH36 α-Galactosidase (TmGalA) Confirms the Mechanistic Commonality of Clan GH-D Glycoside Hydrolases (2007)

Organization of glycoside hydrolase (GH) families into clans expands the utility of information on catalytic mechanisms of member enzymes. This issue was examined for GH27 and GH36 through biochemical analysis of GH36 alpha-galactosidase from Thermotoga maritima (TmGalA). Catalytic residues in TmGalA were inferred through structural homology with GH27 members to facilitate design of site-directed mutants. Product analysis confirmed that the wild type (WT) acted with retention of anomeric stereochemistry, analogous to GH27 enzymes. Conserved acidic residues were confirmed through kinetic analysis of D327G and D387G mutant enzymes, azide rescue, and determination of azide rescue products. Mutation of Asp327 to Gly resulted in a mutant that had a 200-800-fold lower catalytic rate on aryl galactosides relative to the WT enzyme. Azide rescue experiments using the D327G enzyme showed a 30-fold higher catalytic rate compared to without azide. Addition of azide to the reaction resulted in formation of azide beta-d-galactopyranoside, confirming Asp327 as the nucleophilic residue. The Asp387Gly mutation was 1500-fold catalytically slower than the WT enzyme on p-nitrophenyl alpha-d-galactopyranoside. Analysis at different pH values produced a bell-shaped curve of the WT enzyme, but D387G exhibited higher activity with increasing pH. Catalyzed reactions with the D387G mutant in the presence of azide resulted in formation of azide alpha-d-galactopryanoside as the product of a retaining mechanism. These results confirm that Asp387 is the acid/base residue of TmGalA. Furthermore, they show that the biochemical characteristics of GH36 TmGalA are closely related to GH27 enzymes, confirming the mechanistic commonality of clan GH-D members.
Synthesis, Structure Elucidation, and Pharmacological Evaluation of 5‐Methyl‐oxymorphone (= 4,5α‐epoxy‐3,14‐dihydroxy‐5,17‐dimethylmorphinan‐6‐one) (1988)

Abstract Synthesis of 5‐methyl‐oxymorphone ( 3 ) was accomplished by oxidation of 5‐methylthebaine ( 4 ) with performic acid, followed by catalytic hydrogenation and cleavage of the 3‐MeO group. X‐Ray analysis confirmed that the 14‐OH group has, like the one in oxymorphone ( 1 ), β‐orientation. Pharmacological studies in vivo and in vitro showed 3 to possess slightly less opioid agonistic properties than 1 .
Interactions between cyclodextrins and cellular components: Towards greener medical applications? (2016)

In the field of host-guest chemistry, some of the most widely used hosts are probably cyclodextrins (CDs). As CDs are able to increase the water solubility of numerous drugs by inclusion into their hydrophobic cavity, they have been widespread used to develop numerous pharmaceutical formulations. Nevertheless, CDs are also able to interact with endogenous substances that originate from an organism, tissue or cell. These interactions can be useful for a vast array of topics including cholesterol manipulation, treatment of Alzheimer's disease, control of pathogens, etc. In addition, the use of natural CDs offers the great advantage of avoiding or reducing the use of common petroleum-sourced drugs. In this paper, the general features and applications of CDs have been reviewed as well as their interactions with isolated biomolecules leading to the formation of inclusion or exclusion complexes. Finally, some potential medical applications are highlighted throughout several examples.
Coordinate Analysis of Murine Immune Cell Surface Markers and Intracellular Phosphoproteins by Flow Cytometry (2005)

Recently, phosphospecific flow cytometry has emerged as a powerful tool to analyze intracellular signaling events in complex populations of cells because of its ability to simultaneously discriminate cell types based on surface marker expression and measure levels of intracellular phosphoproteins. This has provided novel insights into the cell- and pathway-specific nature of immune signaling. However, we and others have found that the fixation and permeabilization steps necessary for phosphoprotein analysis often negatively affect the resolution of cell types based on surface marker analysis and light scatter characteristics. Therefore, we performed a comprehensive profile of >35 different murine surface marker Abs to understand the effects of fixation and permeabilization on surface Ag staining. Fortuitously, approximately 80% of the Abs tested resolved cell populations of interest, although with decreased separation between positive and negative populations and at very different titers than those used on live cells. The other 20% showed either complete loss of separation between populations or loss of intermediately staining populations. We were able to rescue staining of several of these Ags by performing staining after fixation, but before permeabilization, although with limited fluorophore choices. Scatter characteristics of lymphocytes were well retained, but changed dramatically for monocyte and neutrophil populations. These results compile a comprehensive resource for researchers interested in applying phosphospecific flow cytometry to complex populations of cells while outlining steps necessary to successfully apply new surface marker Abs to this platform.
Hydrodynamics of a Large-scale Mixed-Cell Raceway (MCR): Experimental studies (2007)

The Mixed-cell Raceway (MCR) is a design that intends to combine the best characteristics of circular tanks and linear raceways in a single production system. The conceptual idea is to convert traditional linear raceways into a series of hydraulically separated cells, each of which behaves as an individual circular tank. The MCR can take advantage of the solids removal ability of circular tanks and can be managed as either a partial reuse or intensive recirculation system. This study investigated the hydrodynamics of a large-scale (90 m3) MCR composed of three in-series 5.5 m × 5.5 m mixed-cells (∼1 m water depth). Water velocity measurements of the entire tank were used to generate velocity-magnitude contours and vector plots, investigate the distribution of water velocities, and evaluate the self-cleaning characteristics and related management issues of an MCR. The grand mean of the water velocities of the three MCR in-series cells was 16.5 cm/s (16.1, 15.5, and 17.8 cm/s for cell 1, cell 2, and cell 3, respectively). Results showed that water velocities decreased somewhat in a linear manner from the tank bottom to the top, i.e., 18.9, 15.8 and 14.7 cm/s, and in the same way from the periphery (21.9 cm/s) to the center of the cells (3.7 cm/s). Analyses indicated that these water velocities were in the optimum range to promote fish health and condition as well as to achieve tank self-cleaning. For a water exchange rate of 1.7 volumes per hour and an operating head of 1.36 m in the jet port manifolds, the power requirements of the MCR reached 8.9 W/m3. Contour and vector velocity plots showed that the mixed-cells develop a well-defined rotational pattern around the center drain. Also, strategically located water jets directed across the width of the MCR were able to limit the rotational flow to each cell and create the required counter-rotational pattern between adjacent cells. Velocity vectors showed a relatively low turbulence in the corners of the cells, even in the middle cell (cell 2) that had solid-walls on only two sides. Velocity vectors and contour plots also suggested an absence of dead volumes or short-circuiting within the cells, indicating that adequate mixing was being attained in the MCR.
Communication through virtual active objects overlaid onto the real world (2000)

A SpaceTag is a virtual object that can be accessed only from limited locations and time period. Users of the SpaceTag System can see and create SpaceTags using a portable terminal with a location sensor. We have categorized it as an overlaid virtual system, in which virtual objects are overlaid onto the real world based on time and space coordinates. This architecture is more promising, general purpose, low-cost, and more realistic than other location-aware system architectures like augmented reality that attaches virtual information onto real objects. The SpaceTag System can be offered as a public information service that is a common platform for gaming, advertising, city guide information, etc. We have already implemented a prototype.
Process Modelling and Simulation for 1-Butanol Removing from Fermentation Broth by Extraction with Oleyl Alcohol (2012)

Industrially 1-butanol is a petroleum-based chemical, which can be produced also using renewable biomass resources. 1-Butanol is used as a raw material for the production of bio-based alternatives to many petroleum chemicals, to fossil fuels additivation or, as an alternative fuel. It has higher energy content and lower vapour pressure in the same thermodynamically conditions if compared to methanol and ethanol and also exhibits higher miscibility with diesel fuel and gasoline. These characteristics make 1-butanol a better fuel than lower aliphatic alcohols. In the early 20th century, the fermentation of sugars by Clostridium acetobutylicum was used extensively for the industrial production of a mixture of 1-butanol, acetone, and ethanol. However, high substrate, fermentation and separation costs combined with low yields resulted in the demise of the fermentation process in favour of petrochemical production routes. Due to the present increased interest in finding renewable bio energy resource, recent research efforts have included genetic engineering to increase 1-butanol production, optimizing fermentation and reactor conditions. In the same time, efforts are made in order to find proper techniques for 1-butanol separation and purification. Among the solvents subject to be used in the separation by extraction, oleyl alcohol proved to be one of the most efficient. This alcohol has a rather high cost price but fulfils all the other requirements for a solvent (environment friendly, fire or explosion safe, easily to regenerate and reuse). If the solvent losses during operation are minimized it is possible that the separation process becomes profitable. The present paper presents the results obtained in modelling and simulation of a process unit for 1-butanol separation from the bio fermentation reactor product mixture using oleyl alcohol extraction, results that underlie the economic performance analysis. Process modelling and simulation was realized using Aspen-HYSYS® 7.1.
Biomaterials: defining the mechanical properties of natural tissues and selection of replacement materials (1999)

This article reviews the problems which are encountered in defining the mechanical properties of natural tissues, and in replacing them with synthetic materials in the human body. It describes how death, ageing, degeneration, pathology and individual variability influence the properties of natural tissues. Experimental problems arise from degradation and testing conditions; these are illustrated by the properties of the nucleus pulposus of the intervertebral disc. Replacement of natural tissues by graft materials and the products of tissue engineering is then described. Synthetic replacement materials should be biocompatible, i.e. they should not cause adverse reactions in the human body. However, polymers which hydrolyse in the body fluids may be useful for implants which are intended to have a limited life or for controlled release of drugs. Synthetic implant materials may attempt to mimic natural tissues but there may be a problem of attaching them to the surrounding tissue. Artificial ligaments provide an example of implants of this kind. Total hip replacement is used to illustrate the successful use of conventional engineering materials. Finally, safety issues are described; an implant material must be biocompatible, have the required mechanical strength, be sterile and should be incorporated into a device so that its performance in the living patient can be monitored.
The Enzymatic Function of Tafazzin (2006)

Tafazzin is a putative enzyme that is involved in cardiolipin metabolism, it may carry mutations responsible for Barth syndrome. To identify the biochemical reaction catalyzed by tafazzin, we expressed the full-length isoform of Drosophila melanogaster tafazzin in a baculovirus-Sf9 insect cell system. Tafazzin expression induced a new enzymatic function in Sf9 cell mitochondria, namely 1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine:monolysocardiolipin linoleoyltransferase. We also found evidence for the reverse reaction, because tafazzin expression caused transfer of acyl groups from phospholipids to 1-[14C]palmitoyl-2-lyso-phosphatidylcholine. An affinity-purified tafazzin construct, tagged with the maltose-binding protein, catalyzed both forward and reverse transacylations between cardiolipin and phosphatidylcholine, but was unable to utilize CoA or acyl-CoA as substrates. Whereas tafazzin supported transacylations between various phospholipid-lysophospholipid pairs, it showed the highest rate for the phosphatidylcholine-cardiolipin transacylation. Transacylation activities were about 10-fold higher for linoleoyl groups than for oleoyl groups, and they were negligible for arachidonoyl groups. The data show that Drosophila tafazzin is a CoA-independent, acyl-specific phospholipid transacylase with substrate preference for cardiolipin and phosphatidylcholine.
CS proofs (2002)

This paper puts forward a computationally-based notion of proof and explores its implications to computation at large. In particular, given a random oracle or a suitable cryptographic assumption, we show that every computation possesses a short certificate vouching its correctness, and that, under a cryptographic assumption, any program for a /spl Nscr//spl Pscr/-complete problem is checkable in polynomial time. In addition, our work provides the beginnings of a theory of computational complexity that is based on "individual inputs" rather than languages.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>
Simultaneous determination of guanidinoacetate, creatine and creatinine in urine and plasma by un-derivatized liquid chromatography-tandem mass spectrometry (2008)

A tandem mass spectrometric method has been developed and validated for the underivatized determination of guanidinoacetate, creatine and creatinine in human urine and plasma. Minimal sample preparation coupled with a rapid run time make the method applicable to the routine screening of patients with suspected CCDS.
Controlled/Living Radical Polymerization: Progress in ATRP (2009)

State of the art of Controlled Radical Polymerization (CRP) is presented. Stable free radical polymerization, atom transfer radical polymerization, and degenerate transfer processes, including reversible addition fragmentation transfer are discussed as the most successful CRP techniques. They provide access to new materials from many commercially available monomers under undemanding conditions. Better understanding of the structure-reactivity relationship for the various CRPs and a deeper insight into the mechanistic features can expand range of polymerizable monomers and polymer architectures. Development of a precise relationship between macromolecular structure, processing and final properties of materials will help to find new applications and accelerate commercialization of CRP products.
Molecular staging of bladder cancer with RT-PCR assay for CK20 in peripheral blood, bone marrow and lymph nodes: comparison with standard histological staging. (2006)

Lymph node CK 20 RT-PCR correlates with pathological stage in bladder cancer. The CK20 RT-PCR assay appears to be a highly sensitive and specific method for detecting circulating tumor cells and residual disease in lymph nodes in patients with invasive bladder cancer. Further evaluation of the significance of CK20 as a molecular marker for staging and follow-up in these patients is necessary.
Preneoplastic conditions of the liver. (1996)

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. The majority of patients who develop HCC have underlying cirrhosis, which suggests that cirrhosis itself represents a preneoplastic condition. Nevertheless, whereas patients with cirrhosis of any origin are at increased risk of developing HCC, those with chronic hepatitis B or C infection seem to be at greatest risk. Patients with cirrhosis resulting from chronic alcohol use, hemochromatosis, autoimmune hepatitis, or alpha-1 antitrypsin deficiency have less risk of developing this cancer, and some hepatic diseases, such as primary biliary cirrhosis and Wilson's disease, do not predispose affected persons to an appreciable risk of developing HCC. Certain histological features, such as liver cell dysplasia and macroregenerative nodules, may represent preneoplastic alterations of hepatocytes, but these changes do not seem to be a necessary step in the evolution of liver cancer. The pathogenesis of HCC is unclear, but seems to involve several steps. Hepatitis B virus infection may result in the malignant transformation of hepatocytes by some directly oncogenic mechanism, whereas other necroinflammatory conditions probably predispose to the development of HCC through the introduction of genetic alterations coupled with a reduction of genetic repair functions. Screening patients at risk for the development of HCC using alpha fetoprotein measurements and ultrasonography is widely practiced despite inconclusive evidence of efficacy. If screening is performed, the program used should be tailored to the perceived risk for a particular patient.
The Genesis of Molecular Electronics (2002)

Molecular electronics is, relatively speaking, a young field. Even so, there have been many significant advances and a much greater understanding of the types of materials that will be useful in molecular electronics, and their properties. The purpose of this review is to provide a broad basis for understanding the areas where new advances might arise, and to provide introduction to the subdisciplines of molecular electronics. This review is divided into two major parts; an historical examination of the development of conventional electronics, which should provide some understanding of the push towards molecular electronics. The problems associated with continuing to shrink conventional systems are presented, along with references to some of the efforts to solve them. This section is followed by an in-depth look at the most important research into the types of behaviors that molecular systems have been found to display.
Efficient Intracellular Delivery of an Exogenous Protein GFP with Genetically Fused Basic Oligopeptides (2001)

Several oligopeptides, derived from certain proteins, translocate as a form fused to small molecules or exogenous proteins across the plasma membrane into cells. Some of these oligopeptides, the so-called protein-transduction domains (PTDs), contain a high proportion of basic residues. The translocation of some of these basic PTDs, such as oligoarginines, has been studied as chemically fused forms to other organic compounds. In this study, we also tested to determine whether or not oligoarginines, when fused genetically to an exogenous protein such as GFP, are also able to translocate efficiently across the plasma membrane. The oligoarginine Rn (n = 5,6,7,8,9)-GFP fusion proteins were translocated quite efficiently, and the transduction efficiency increased in proportion to the number of arginine residues. However, the cellular uptake of the oligolysine-GFP fusion proteins was less efficient than that of the corresponding oligoarginine-GFP fusion proteins. When fused to GFP, the translocation efficiency of R5 was similar to that of Tat(49-57)(RKKRRQRRR). This finding suggests that the arginine homo-oligopeptide is more efficient than other PTDs which contain a mixture of basic residues. On the other hand, both the K9- and Tat(49-57)-GFP fusion proteins were transduced with similar efficiencies. It appears that basic oligopeptides may be useful for the efficient translocation of diverse exogenous proteins as genetically fused forms.
Interaction with Both Domain I and III of Albumin Is Required for Optimal pH-dependent Binding to the Neonatal Fc Receptor (FcRn) (2014)

Albumin is an abundant blood protein that acts as a transporter of a plethora of small molecules like fatty acids, hormones, toxins, and drugs. In addition, it has an unusual long serum half-life in humans of nearly 3 weeks, which is attributed to its interaction with the neonatal Fc receptor (FcRn). FcRn protects albumin from intracellular degradation via a pH-dependent cellular recycling mechanism. To understand how FcRn impacts the role of albumin as a distributor, it is of importance to unravel the structural mechanism that determines pH-dependent binding. Here, we show that although the C-terminal domain III (DIII) of human serum albumin (HSA) contains the principal binding site, the N-terminal domain I (DI) is important for optimal FcRn binding. Specifically, structural inspection of human FcRn (hFcRn) in complex with HSA revealed that two exposed loops of DI were in proximity with the receptor. To investigate to what extent these contacts affected hFcRn binding, we targeted selected amino acid residues of the loops by mutagenesis. Screening by in vitro interaction assays revealed that several of the engineered HSA variants showed decreased binding to hFcRn, which was also the case for two missense variants with mutations within these loops. In addition, four of the variants showed improved binding. Our findings demonstrate that both DI and DIII are required for optimal binding to FcRn, which has implications for our understanding of the FcRn-albumin relationship and how albumin acts as a distributor. Such knowledge may inspire development of novel HSA-based diagnostics and therapeutics.
A widely expressed human protein-tyrosine phosphatase containing src homology 2 domains. (1993)

A cDNA encoding a nontransmembrane protein-tyrosine phosphatase (PTP; EC 3.1.3.48), termed PTP2C, was isolated from a human umbilical cord cDNA library. The enzyme contains a single phosphatase domain and two adjacent copies of the src homology 2 (SH2) domain at its amino terminus. A variant of PTP2C (PTP2Ci) which has four extra amino acid residues within the catalytic domain has been identified also. PTP2C is widely expressed in human tissues and is particularly abundant in heart, brain, and skeletal muscle. The catalytic domain of PTP2C was expressed as a recombinant enzyme in Escherichia coli and purified to near homogeneity by two chromatographic steps. The recombinant enzyme was totally specific toward phosphotyrosine residues. The structural similarity between PTP2C and the previously described PTP1C suggests the existence of a subfamily of SH2-containing PTPs; these may play an important role in signal transduction through interaction of their SH2 domains with phosphotyrosine-containing proteins.