gebhart/patrepeval huggingface dataset:
Design and numerical simulation of a DNA electrophoretic stretching device (2006)

DNA stretching is now a key technology in emerging DNA mapping devices such as direct linear analysis (DLA), though DNA stretching in a high throughput manner is still a challenging problem. In this work, we present a new microfluidic channel design to enhance DNA stretching using kinematic analysis and Brownian dynamics-finite element method (BD-FEM). Our group recently showed in experiments that the extensional electrophoretic field arising from a hyperbolic microcontraction can be utilized to stretch T4-DNA. We demonstrate the reliability of our BD-FEM model for the present problem by showing that the numerical predictions are consistent with the experimental data for the hyperbolic channel. We then investigate DNA stretching for four different funnel shapes. Surprisingly the maximum mean DNA stretch is quite similar in all four designs. Finally, we propose a new design with a side-feeding branch to enhance stretching based on a kinematic analysis along different feeding locations. Our numerical simulation predicted that DNA stretching can be dramatically enhanced using side-feeding.
DNA replication meets genetic exchange: Chromosomal damage and its repair by homologous recombination (2001)

Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair.
Distribution Volume Ratios without Blood Sampling from Graphical Analysis of PET Data (1996)

The distribution volume ratio (DVR), which is a linear function of receptor availability, is widely used as a model parameter in imaging studies. The DVR corresponds to the ratio of the DV of a receptor-containing region to a nonreceptor region and generally requires the measurement of an arterial input function. Here we propose a graphical method for determining the DVR that does not require blood sampling. This method uses data from a nonreceptor region with an average tissue-to-plasma efflux constant k2 to approximate the plasma integral. Data from positron emission tomography studies with [11C]raclopride (n = 20) and [11C]d-threo-methylphenidate ([11C]dMP) (n = 8) in which plasma data were taken and used to compare results from two graphical methods, one that uses plasma data and one that does not. k2 was 0.163 and 0.051 min-1 for [11C]raclopride and [11C]dMP, respectively. Results from both methods were very similar, and the average percentage difference between the methods was -0.11% for [11C]raclopride and 0.46% for [11C]dMP for DVR of basal ganglia (BG) to cerebellum (CB). Good agreement between the two methods was also achieved for DVR images created by both methods. This technique provides an alternative method of analysis not requiring blood sampling that gives equivalent results for the two ligands studied. It requires initial studies with blood sampling to determine the average kinetic constant and to test applicability. In some cases, it may be possible to neglect the k2 term if the BG/CB ratio becomes reasonably constant for a sufficiently long period of time over the course of the experiment.
Crystal Structures of the Short-Chain Flavin Reductase HpaC from Sulfolobus tokodaii Strain 7 in Its Three States:  NAD(P)+-Free, NAD+-Bound, and NADP+-Bound, (2006)

4-Hydroxyphenylacetate (4-HPA) is oxidized as an energy source by two component enzymes, the large component (HpaB) and the small component (HpaC). HpaB is a 4-HPA monooxygenase that utilizes FADH(2) supplied by a flavin reductase HpaC. We determined the crystal structure of HpaC (ST0723) from the aerobic thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 in its three states [NAD(P)(+)-free, NAD(+)-bound, and NADP(+)-bound]. HpaC exists as a homodimer, and each monomer was found to contain an FMN. HpaC preferred FMN to FAD because there was not enough space to accommodate the AMP moiety of FAD in its flavin-binding site. The most striking difference between the NAD(P)(+)-free and the NAD(+)/NADP(+)-bound structures was observed in the N-terminal helix. The N-terminal helices in the NAD(+)/NADP(+)-bound structures rotated ca. 20 degrees relative to the NAD(P)(+)-free structure. The bound NAD(+) has a compact folded conformation with nearly parallel stacking rings of nicotinamide and adenine. The nicotinamide of NAD(+) stacked the isoalloxazine ring of FMN so that NADH could directly transfer hydride. The bound NADP(+) also had a compact conformation but was bound in a reverse direction, which was not suitable for hydride transfer.
A single‐point mutation enables lactate dehydrogenase from Bacillus subtilis to utilize NAD+ and NADP+ as cofactor (2011)

Abstract The NAD + ‐dependent lactate dehydrogenase from Bacillus subtilis (BsLDH) catalyzes the enantioselective reduction of pyruvate to lactate. BsLDH is highly specific to NAD + and exhibits only a low activity with NADP + as cofactor. Based on the high activity and good stability of LDHs, these enzymes have been frequently used for the regeneration of NAD + . While an application in the regeneration of NADP + is not sufficient due to the cofactor preference of the BsLDH. In addition, NADP + ‐dependent LDHs have not yet been found in nature. Therefore, a structure‐based approach was performed to predict amino acids involved in the cofactor specificity. Methods of site‐saturation mutagenesis were applied to vary these amino acids, with the aim to alter the cofactor specificity of the BsLDH. Five constructed libraries were screened for improved NADP + acceptance. The mutant V39R was identified to have increased activity with NADP + relative to the wild type. V39R was purified and biochemically characterized. V39R showed excellent kinetic properties with NADP(H) and NAD(H), for instance the maximal specific activity with NADPH was enhanced 100‐fold to 90.8 U/mg. Furthermore, a 249‐fold increased catalytic efficiency was observed. Surprisingly, the activity with NADH was also significantly improved. Overall, we were able to successfully apply V39R in the regeneration of NADP + in an enzyme‐coupled approach combined with the NADP + ‐dependent alcohol dehydrogenase from Lactobacillus kefir . We demonstrate for the first time an application of an LDH in the regeneration of NADP + .
Predictive value of mucosal TNF-α transcripts in steroid-refractory Crohnʼs disease patients receiving intensive immunosuppressive therapy (2006)

Quantification of mucosal TNF-alpha transcripts prior to therapy allows identification of patients achieving long-term remission upon immunosuppression with infliximab or cyclophosphamide. Real-time PCR might have considerable potential in the analysis of disease activity and subsequent clinical management of patients with immunosuppressive therapies.
Cloning of Amadoriase I Isoenzyme from Aspergillus sp.:  Evidence of FAD Covalently Linked to Cys342, (2000)

Amadoriases are a novel class of FAD enzymes which catalyze the oxidative deglycation of glycated amino acids to yield corresponding amino acids, glucosone, and H(2)O(2). We previously reported the purification and characterization of two amadoriase isoenzymes from Aspergillus fumigatus and the molecular cloning of amadoriase II. To identify the primary structure of amadoriase I, we prepared a cDNA library from Aspergillus fumigatus and isolated a clone using a probe amplified by polymerase chain reaction with primers designed according to the partial amino acid sequences from peptide mapping. The primary structure of the enzyme deduced from the nucleotide sequence comprises 445 amino acid residues. The enzyme contains 1 mol of FAD as a cofactor, which is covalently linked to Cys342, as determined by mutagenesis analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and electrospray ionization-collisional-activated dissociation tandem mass spectrometry. Sequence alignment studies show that amadoriase I has 22% homology with monomeric sarcosine oxidase in which FAD is also linked to a homologous Cys residue. Amadoriases are of potential importance as tools for uncoupling hyperglycemia and glycation reactions that are thought to play a role in diabetic complications.
Human tumor infiltrating lymphocytes. Analysis of lymphokine mRNA expression and relevance to cancer immunotherapy. (1989)

Tumor infiltrating lymphocytes (TIL) isolated from 12 patients with metastatic malignant melanoma, renal cell carcinoma, or breast adenocarcinoma were expanded in rIL-2 for 22 to 45 days (median 33 days) and analyzed for lymphokine mRNA expression and patterns of TCR gene rearrangement. All TIL cultures were significantly enriched for T cells, with CD3+ CD8+ cells predominant in 8 of 10 cases tested, and demonstrated an oligoclonal (rather than polyclonal) pattern of TCR gene rearrangement. Nine of 12 cultures could effectively lyse the autologous targets in short term chromium release assays. IL-2 expanded-TIL expressed mRNA for TNF-alpha and TNF-beta (lymphotoxin) and, in 5 of 9 (41%) cases, granulocyte/macrophage-colony stimulating factor mRNA but not IL-1 beta or IL-2 transcripts. Cultured TIL deprived of rIL-2 for 4 days did not constitutively express mRNA for any of the lymphokines tested. One long term TIL line in culture was followed and periodically tested for lytic activity and TNF-mRNA expression. Loss of the specific cytolytic but not proliferative activity at day 85 was associated with disappearance of TNF mRNA. Profiles of lymphokine secretion may provide a useful marker for functionally characterizing different T cell subsets and may provide correlates of the in vivo anti-tumor effects of these cells when TIL are adoptively transferred into cancer-bearing patients.
Thirty Years of Water and Environmental Economics (2000)

The plan for these essays, supplied by our editor, suggests beginning with a paragraph on how each author got in to the water field. He says th at in his case it was “a flu ke.” So it was in m ine. I wen t to graduate sc hool aim ing to become a development economist, based on what I had seen and learned in nine months or so living in Uganda and working as an Research Assistant (R.A.) for an econom ist who was doing a book on the East African economy as it then was. To make a long, and to me painful, story short, I had after my second year determined that I would not be able to find a thesis advisor from among the several Harvard development econo mists. In the midst of the resulting depression I was asked to lunch by Henry Jacoby, then running the Harvard Water Program for Bob Dorfman and Harold Thomas. He was looking for a graduate student on whom to lavish some suppor t. It took abo ut five minutes for me to see the
The more the merrier: high-throughput single-molecule techniques (2017)

The single-molecule approach seeks to understand molecular mechanisms by observing biomolecular processes at the level of individual molecules. These methods have led to a developing understanding that for many processes, a diversity of behaviours will be observed, representing a multitude of pathways. This realisation necessitates that an adequate number of observations are recorded to fully characterise this diversity. The requirement for large numbers of observations to adequately sample distributions, subpopulations, and rare events presents a significant challenge for single-molecule techniques, which by their nature do not typically provide very high throughput. This review will discuss many developing techniques which address this issue by combining nanolithographic approaches, such as zero-mode waveguides and DNA curtains, with single-molecule fluorescence microscopy, and by drastically increasing throughput of force-based approaches such as magnetic tweezers and laminar-flow techniques. These methods not only allow the collection of large volumes of single-molecule data in single experiments, but have also made improvements to ease-of-use, accessibility, and automation of data analysis.
The Experimental Integrated Switched Network - a System-Level Network Test Facility (1983)

An Experimental Integrated Switched Network (EISN) has been developed to provide a system-level testbed for the evaluation of advanced communications networking techniques, including survivable network routing algorithms using a mix of transmission media, for application in the Defense Switched Network (DSN). EISN includes five CONUS sites linked by a wideband demand-assigned satellite channel and by dialed-up terrestrial trunks for alternate satellite/terrestrial routing experiments. Experiments to date have validated techniques for integration of circuit-switched terrestrial systems with the demand-assigned satellite system, and for the establishment of alternate routes over satellite and terrestrial paths. Currently, candidate routing algorithms for application in the DSN are being implemented and tested using external routing/controller processors attached to digital circuit switches at EISN sites. In addition, EISN is also being used to support data communication experiments using DoD standard data protocols in a combined satellite/ terrestrial network environment. Work is ongoing both in system experiments and in testbed developments to include additional capabilities. This paper represents a description and status report on both the testbed and the experimental efforts.
Taql RFLPs at the Wilms' tumor gene (WT1) (1991)

Clone WT33 contains a 2.3 kb cDNA of one of the candidate genes for Wilms' tumor at lI p13 (1). A 1.8 kb EcoRI fragment of the plasmid was used as a probe, which covered the 5' portion of the gene. Polymorphisms: TaqI detected two 2-allele polymorphisms (set Aa and Ab) with fragment size of 3.4 kb (Al) or 3.2 kb (A2), and 2.2 kb (Al) or 1.5 kb (A2). Constant bands of 6.6, 3.7, 1.7, 1.2 and 0.8 kb were also detected. Frequencies: Studied in 65 unrelated individuals of the normal Japanese (2). Frequencies of haplotype were estimated with the aid of tumor DNAs with loss of heterozygosity.
Neutrophil transendothelial migration is independent of tight junctions and occurs preferentially at tricellular corners (1997)

Since macromolecular permeability between endothelial cells is regulated by tight junctions (zonula occludens), we wished to determine whether they also regulate neutrophil transendothelial migration. HUVEC monolayers, a commonly used model for studying leukocyte transmigration, were characterized using electric cell substrate impedance sensing and transmission electron microscopy. We show that culture medium containing endothelial cell growth supplement (50 microg/ml) was sufficient and necessary for the development of endothelial tight junctions. The frequency with which tight junctions were observed by transmission electron microscopy was further increased (twofold) by culturing HUVEC monolayers in a 1:1 mixture of endothelial medium and astrocyte-conditioned medium. These astrocyte-conditioned HUVEC monolayers showed a >1.5-fold increase in transcellular electrical resistance. The extent of neutrophil migration across IL-1-treated (10 U/ml for 4 h) HUVEC monolayers was the same whether tight junctions were present or absent, and the molecular requirements for neutrophil transmigration (CD18 and intercellular adhesion molecule-1) were unaffected by culturing in astrocyte-conditioned medium. Immunostaining for proteins associated with the intercellular junctional domain (occludin, ZO-1, cadherin, beta-catenin, gamma-catenin, and platelet-endothelial cell adhesion molecule-1) was localized to the endothelial borders, regardless of the culture conditions. Discontinuities were observed in the border staining for occludin, ZO-1, cadherin, and beta-catenin at the tricellular corner where the borders of three endothelial cells intersected. Significantly, 75% of neutrophil migration across IL-1-treated HUVEC monolayers occurred at tricellular corners. It appears that neutrophils preferentially migrate around endothelial tight junctions by crossing at tricellular corners rather than passing through the tight junctions that lie between two endothelial cells.
Quantifying effect of statins on low density lipoprotein cholesterol, ischaemic heart disease, and stroke: systematic review and meta-analysis (2003)

Statins can lower LDL cholesterol concentration by an average of 1.8 mmol/l which reduces the risk of IHD events by about 60% and stroke by 17%.
Transcriptional and Proteomic Profiles of Group B Streptococcus Type V Reveal Potential Adherence Proteins Associated with High-Level Invasion (2007)

Group B Streptococcus (GBS) is an opportunistic organism that can harmlessly colonize the human gut, vagina, and rectum but can also cause pneumonia, sepsis, and meningitis in neonates born to colonized mothers. We have shown previously that growth rate and oxygen level regulate the ability of GBS to invade eukaryotic cells in vitro. Herein we extend and expand on these observations to show that GBS type V, an emergent serotype, grown in a chemostat at a cell mass-doubling time (t(d)) of 1.8 h with oxygen invaded human ME-180 cervical epithelial cells in large numbers compared with those grown at the same t(d) without oxygen or at a slower t(d) of 11.0 h. The fact that several GBS type V cell wall-associated and membrane proteins were expressed exclusively under the invasive growth condition prompted an investigation, using genomics and proteomics, of all upregulated genes and proteins. Several proteins with potential roles in adherence were identified, including an undefined surface antigen (SAG1350), a lipoprotein (SAG0971), penicillin-binding protein 2b (SAG0765), glyceraldehyde-3-phosphate dehydrogenase (SAG0823), and an iron-binding protein (SAG1007). Mouse antisera to these five proteins inhibited binding of GBS type V to ME-180 cells by > or =85%. Recombinant undefined surface antigen (SAG1350), lipoprotein (SAG0971), and penicillin-binding protein 2b (SAG0765) each bound to ME-180 cells in a dose-dependent fashion, confirming their ability to act as ligands. Collectively, these data increase the number of potential GBS adherence factors and also suggest a role for these surface-associated proteins in initial pathogenic events.
Phospholipase C‐β and ovarian sex steroids in pig granulosa cells (1999)

We compared the membrane effects of estradiol, progesterone, and androstenedione in a single experimental model, the ovarian granulosa cells collected from immature Large White sows. We measured changes in cytosolic free calcium concentration ([Ca2+]i) in confluent Fura-2 loaded cells. We used pharmacological tools and polyclonal phospholipase C-β (PLC-β) antibodies. Each steroid (0.1 pM to 1 nM) transiently increased intracellular calcium concentration ([Ca2+]i) within 5 sec. They mobilized Ca2+ from the endoplasmic reticulum as shown by using two phospholipase C inhibitors, neomycin and U-73122. Ca2+ mobilization involved PLC-β1 for progesterone, PLC-β2 for estradiol and PLC-β4 for androstenedione. A pertussis toxin-insensitive G protein was involved in the effects of progesterone on Ca2+ mobilization whereas estradiol and androstenedione effects were mediated via a pertussis toxin-sensitive G-protein. Ca2+ influx from the extracellular milieu was involved in the increase in [Ca2+]i induced by progesterone and estradiol, but not by androstenedione. Influx of Ca2+ was independent of Ca2+ mobilization from calcium stores, and it was suggested that L-type Ca2+ channels for estradiol and T-type Ca2+ channels for progesterone were involved. The three steroids had no effect on cAMP. Rapid effects of progesterone, estradiol, and androstenedione involved a direct action on cell membrane elements such as PLC-β, G-proteins, and calcium channels, and these mechanisms were hormone-specific. J. Cell. Biochem. 74:50–60, 1999. © 1999 Wiley-Liss, Inc.
The PI3K/Akt and mTOR/P70S6K Signaling Pathways in Human Uveal Melanoma Cells: Interaction with B-Raf/ERK (2010)

Activated PI3K/Akt attenuates the inhibitory effects of rapamycin on cell proliferation and thus serves as a negative feedback mechanism. This finding suggests that rapamycin is unlikely to inhibit uveal melanoma growth. In contrast, targeting PI3K while inhibiting B-Raf/ERK may be a promising approach to reduce the proliferation of uveal melanoma cells.
Proliferation and maturation of human erythroid progenitors in liquid culture (1989)

Hemopoiesis is studied in vitro mainly in semisolid culture, where hemopoietic progenitors develop into discrete colonies. We describe a liquid culture system that supports the proliferation and maturation of human erythroid progenitors. We seeded mononuclear cells from the peripheral blood (PB) of patients with beta-thalassemia in liquid medium in the presence of conditioned medium from human bladder carcinoma cells. Seven days later, RBCs, normoblasts, granulocytes, and monocytes disappeared, and the number of lymphocytes dropped considerably. In contrast, erythroid colony-forming cells increased fourfold to tenfold. The next step entailed the removal of colony-stimulating factor (CSF) and CSF-secreting cells, the exclusion of macrophages by harvesting nonadherent cells, and the lysis of T lymphocytes by treatment with monoclonal rat antihuman lymphocyte antibodies (CAMPATH-1) and complement. Reculture of the remaining cells in liquid medium supplemented with recombinant erythropoietin (EPO) resulted in the exclusive development of erythroid cells, with myeloid cells reduced to less than 2%. Stainable hemoglobin (Hb) appeared on day 3, with over 85% of the population containing hemoglobin by day 11 and the cell number increasing from 0.2 X 10(6) to 3 X 10(6) mL. By permitting the manipulation of culture conditions and components and increasing the cell yield, the liquid system may facilitate quantitative analysis of growth kinetics as well as biochemical and immunologic characterization of the developing erythroid cell.