gebhart/patrepeval huggingface dataset:
Involvement ofDFNB59 mutations in autosomal recessive nonsyndromic hearing impairment (2007)

In a consanguineous Turkish family, a locus for autosomal recessive nonsyndromic hearing impairment (ARNSHI) was mapped to chromosome 2q31.1-2q33.1. Microsatellite marker analysis in the complete family determined the critical linkage interval that overlapped with DFNB27, for which the causative gene has not yet been identified, and DFNB59, a recently described auditory neuropathy caused by missense mutations in the DFNB59 gene. The 352-amino acid (aa) DFNB59 gene product pejvakin is present in hair cells, supporting cells, spiral ganglion cells, and the first three relays of the afferent auditory pathway. A novel homozygous nonsense mutation (c.499C>T; p.R167X) was detected in the DFNB59 gene, segregating with the deafness in the family. The mRNA derived from the mutant allele was found not to be degraded in lymphocytes, indicating that a truncated pejvakin protein of 166 aa may be present in the affected individuals. Screening of 67 index patients from additional consanguineous Turkish families with autosomal recessive hearing impairment revealed a homozygous missense mutation (c.547C>T; p.R183W) that segregates with the hearing impairment in one family. Furthermore, in a panel of 83 Dutch patients, two additional novel mutations (c.509_512delCACT; p.S170CfsX35 and c.731T>G; p.L244R), which were not present in ethnically matched controls, were found heterozygously. Together, our data indicate that also nonsense mutations in DFNB59 cause nonsyndromic hearing loss, but that mutations in DFNB59 are not a major cause of nonsyndromic hearing impairment in the Turkish and Dutch population.
Evaluating acellular versus cellular perfusate composition during prolongedex vivolung perfusion after initial cold ischaemia for 24 hours (2015)

Normothermic ex vivo lung perfusion (EVLP) has developed as a powerful technique to evaluate particularly marginal donor lungs prior to transplantation. In this study, acellular and cellular perfusate compositions were compared in an identical experimental setting as no consensus has been reached on a preferred technique yet. Porcine lungs underwent EVLP for 12 h on the basis of an acellular or a cellular perfusate composition after 24 h of cold ischaemia as defined organ stress. During perfusion, haemodynamic and respiratory parameters were monitored. After EVLP, the lung condition was assessed by light and transmission electron microscopy. Aerodynamic parameters did not show significant differences between groups and remained within the in vivo range during EVLP. Mean oxygenation indices were 491 ± 39 in the acellular group and 513 ± 53 in the cellular group. Groups only differed significantly in terms of higher pulmonary artery pressure and vascular resistance in the cellular group. Lung histology and ultrastructure were largely well preserved after prolonged EVLP and showed only minor structural alterations which were similarly present in both groups. Prolonged acellular and cellular EVLP for 12 h are both feasible with lungs prechallenged by ischaemic organ stress. Physiological and ultrastructural analysis showed no superiority of either acellular or cellular perfusate composition.
Assessment of the Effects of MPTP and Paraquat on Dopaminergic Neurons and Microglia in the Substantia Nigra Pars Compacta of C57BL/6 Mice (2016)

The neurotoxicity of paraquat dichloride (PQ) was assessed in two inbred strains of 9- or 16-week old male C57BL/6 mice housed in two different laboratories and compared to the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). PQ was administered by intraperitoneal injections; either once (20 mg/kg) or twice (10 mg/kg) weekly for 3 weeks, while MPTP-HCl was injected 4 times on a single day (20 mg/kg/dose). Brains were collected 8, 16, 24, 48, 96 or 168 hours after the last PQ treatment, and 48 or 168 hours after MPTP treatment. Dopamine neurons in the substantia nigra pars compacta (SNpc) were identified by antibodies to tyrosine hydroxylase (TH+) and microglia were identified using Iba-1 immunoreactivity. The total number of TH+ neurons and the number of resting and activated microglia in the SNpc at 168 hours after the last dose were estimated using model- or design-based stereology, with investigators blinded to treatment. In a further analysis, a pathologist, also blinded to treatment, evaluated the SNpc and/or striatum for loss of TH+ neurons (SNpc) or terminals (striatum), cell death (as indicated by amino cupric silver uptake, TUNEL and/or caspase 3 staining) and neuroinflammation (as indicated by Iba-1 and/or GFAP staining). PQ, administered either once or twice weekly to 9- or 16-week old mice from two suppliers, had no effect on the number of TH+ neurons or microglia in the SNpc, as assessed by two groups, each blinded to treatment, using different stereological methods. PQ did not induce neuronal cell loss or degeneration in the SNpc or striatum. Additionally, there was no evidence of apoptosis, microgliosis or astrogliosis. In MPTP-treated mice, the number of TH+ neurons in the SNpc was significantly decreased and the number of activated microglia increased. Histopathological assessment found degenerating neurons/terminals in the SNpc and striatum but no evidence of apoptotic cell death. MPTP activated microglia in the SNpc and increased the number of astrocytes in the SNpc and striatum.
Induction of cytochrome P-450 by glucocorticoids in rat liver. I. Evidence that glucocorticoids and pregnenolone 16 alpha-carbonitrile regulate de novo synthesis of a common form of cytochrome P-450 in cultures of adult rat hepatocytes and in the liver in vivo. (1984)

We administered a series of steroid hormones to primary nonproliferating cultures of adult rat hepatocytes and found that dexamethasone and other glucocorticoids but not sex steroid hormones, mineralocorticoids, or derivatives of pregnenolone other than pregnenolone 16 alpha-carbonitrile (PCN) stimulated de novo synthesis of an immunoreactive protein, indistinguishable from the form of cytochrome P-450 (P450PCN) induced by PCN in rat liver. No difference were discerned among purified liver cytochromes from rats treated with dexamethasone, PCN or dexamethasone plus PCN, among proteolytic digests of these proteins, or among the immunoprecipitated cytochromes prepared from cultured hepatocytes treated with these steroids as judged by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate followed by immunoblot analysis. Of the steroids tested, dexamethasone proved to be the most efficacious inducer increasing the rate of synthesis of P450PCN from 0.05% of total cellular protein synthesis in incubated control cultures (measured as incorporation of [3H]leucine into immunoprecipitable P450PCN) to as much as 9.4% in cultures incubated for 5 days in medium containing dexamethasone (10(-5) M). As with traditional glucocorticoid-responsive liver functions, induction of immunoreactive P450PCN was dependent on the concentration of dexamethasone (10(-8) to 10(-5) M) and was promptly reversed upon withdrawal of the steroid. However, during the 24-h interval between 24 to 48 h of culture age the hepatocytes were refractory to either induction or de-induction of immunoreactive P450PCN even though continuous exposure of the cells to dexamethasone (including this interval) was mandatory for maximal induction of P450PCN at 120 h in culture. Unlike cultured rat hepatocytes, HTC hepatoma cultures failed to exhibit dexamethasone-responsive expression of immunoreactive P450PCN. We conclude that glucocorticoids and PCN constitute a specific "class" of synthetic and endogenous inducers of a single form of cytochrome P-450.
Sequence analysis andin vivoexpression show that alternative splicing of ED-B and ED-A regions of the human fibronectin gene are independent events (1988)

The structure of two alternatively spliced regions. ED-A and ED-B, of human fibronectin gene, was determined, in order to show whether any similarity was present between the two. Although some interesting features are present in each, no obvious common structure or sequence homology was found. Functional analysis of the alternative splicing events was carried out by transient expression in Hela cells. A hybrid gene was constructed by inserting the ED-B region into the third exon of the human alpha 1-globin gene. The transfected hybrid gene is expressed and produces, in Hela cells, two alternatively spliced RNAs, showing a pattern very similar to that observed for the endogenous fibronectin gene in fibroblasts. Cotransfection of this gene with a similar gene containing the ED-A region, shows that no interference is present between the two alternative splicing processes.
Ligand Binding: Molecular Mechanics Calculation of the Streptavidin-Biotin Rupture Force (1996)

The force required to rupture the streptavidin-biotin complex was calculated here by computer simulations. The computed force agrees well with that obtained by recent single molecule atomic force microscope experiments. These simulations suggest a detailed multiple-pathway rupture mechanism involving five major unbinding steps. Binding forces and specificity are attributed to a hydrogen bond network between the biotin ligand and residues within the binding pocket of streptavidin. During rupture, additional water bridges substantially enhance the stability of the complex and even dominate the binding interactions. In contrast, steric restraints do not appear to contribute to the binding forces, although conformational motions were observed.
Characterization of Variable Regions of Monoclonal Antibodies by Top-Down Mass Spectrometry (2007)

A technique for rapid characterization of variable regions of monoclonal antibodies (mAb) is described. Several intact mAbs were analyzed on a Thermo-Fisher LTQ-Orbitrap high-resolution mass spectrometer (MS) by in-source fragmentation. In-source fragmentation has the unique advantage of fragmenting all charge states of a protein at the same time and, thus, greatly improves the sensitivity of the fragment ions over a true MS/MS experiment, where a single charge state is isolated and fragmented. In addition, immediate fragmentation of the protein before tertiary structure formation may also facilitate protein fragmentation. This technique has been proved very useful for top-down analysis of large proteins. In-source fragmentation of mAbs generated a series of fragment ions. In addition to some small b and y ions from the light chain and heavy chain in the low m/z region, a series of b ions corresponding to N-terminal 106-120 residues of both heavy chain and light chain were observed. The cleavage sites for these b ions happen to be near the linker regions between the variable domains and the constant domains of these antibodies. These b ions, therefore, correspond to the entire variable region of each chain. Similar results were obtained for all mAbs analyzed, including both immunoglobulin G1 and G2 molecules. To further characterize the variable regions, these b ions were isolated and fragmented by collision-induced dissociation in the linear trap, followed by mass analysis in the orbitrap. Large number of product ions was observed from these b ions. Many of these product ions are internal fragments between the two disulfide-linked cysteine residues. To demonstrate the capability of the technique, several mAbs were force-oxidized by treating with tert-butyl hydroperoxide, followed by mass spectrometric analysis. In-source fragmentation and MS/MS of the variable region b ions clearly identified the locations of the oxidized methionine.
Improved NADPH supply for xylitol production by engineered Escherichia coli with glycolytic mutations (2011)

Escherichia coli engineered to uptake xylose while metabolizing glucose was previously shown to produce high levels of xylitol from a mixture of glucose and xylose when expressing NADPH-dependent xylose reductase from Candida boidinii (CbXR) (Cirino et al., Biotechnol Bioeng. 2006;95:1167-1176). We then described the effects of deletions of key metabolic pathways (e.g., Embden-Meyerhof-Parnas and pentose phosphate pathway) and reactions (e.g., transhydrogenase and NADH dehydrogenase) on resting-cell xylitol yield (Y RPG: moles of xylitol produced per mole of glucose consumed) (Chin et al., Biotechnol Bioeng. 2009;102:209-220). These prior results demonstrated the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions. This study describes strain modifications that improve coupling between glucose catabolism (oxidation) and xylose reduction using two fundamentally different strategies. We first examined the effects of deleting the phosphofructokinase (pfk) gene(s) on growth-uncoupled xylitol production and found that deleting both pfkA and sthA (encoding the E. coli-soluble transhydrogenase) improved the xylitol Y RPG from 3.4 ± 0.6 to 5.4 ± 0.4. The second strategy focused on coupling aerobic growth on glucose to xylitol production by deleting pgi (encoding phosphoglucose isomerase) and sthA. Impaired growth due to imbalanced NADPH metabolism (Sauer et al., J Biol Chem. 2004;279:6613-6619) was alleviated upon expressing CbXR, resulting in xylitol production similar to that of the growth-uncoupled precursor strains but with much less acetate secretion and more efficient utilization of glucose. Intracellular nicotinamide cofactor levels were also quantified, and the magnitude of the change in the NADPH/NADP+ ratio measured from cells consuming glucose in the absence vs. presence of xylose showed a strong correlation to the resulting Y RPG.
A System for Studying Epithelial-Stromal Interactions Reveals Distinct Inductive Abilities of Stromal Cells from Benign Prostatic Hyperplasia and Prostate Cancer (2004)

The development of normal and abnormal glandular structures in the prostate is controlled at the endocrine and paracrine levels by reciprocal interactions between epithelium and stroma. To study these processes, it is useful to have an efficient method of tissue acquisition for reproducible isolation of cells from defined histologies. Here we assessed the utility of a standardized system for acquisition and growth of prostatic cells from different regions of the prostate with different pathologies, and we compared the abilities of stromal cells from normal peripheral zone, benign prostatic hyperplasia (BPH-S), and cancer to induce the growth of a human prostatic epithelial cell line (BPH-1) in vivo. Using the tissue recombination method, we showed that grafting stromal cells (from any histology) alone or BPH-1 epithelial cells alone produced no visible grafts. Recombining stromal cells from normal peripheral zone with BPH-1 cells also produced no visible grafts (n = 15). Recombining BPH-S with BPH-1 cells generated small, well-organized, and sharply demarcated grafts approximately 3-4 mm in diameter (n = 9), demonstrating a moderate inductive ability of BPH-S. Recombining stromal cells from cancer with BPH-1 cells generated highly disorganized grafts that completely surrounded the host kidney and invaded into adjacent renal tissue, demonstrating induction of an aggressive phenotype. We conclude that acquisition of tissue from toluidine blue dye-stained specimens is an efficient method to generate high-quality epithelial and/or stromal cultures. Stromal cells derived by this method from areas of BPH and cancer induce epithelial cell growth in vivo, which mimics the natural history of these diseases.
Heat Shock Response and Protein Degradation: Regulation of HSF2 by the Ubiquitin-Proteasome Pathway (1998)

Mammalian cells coexpress a family of heat shock factors (HSFs) whose activities are regulated by diverse stress conditions to coordinate the inducible expression of heat shock genes. Distinct from HSF1, which is expressed ubiquitously and activated by heat shock and other stresses that result in the appearance of nonnative proteins, the stress signal for HSF2 has not been identified. HSF2 activity has been associated with development and differentiation, and the activation properties of HSF2 have been characterized in hemin-treated human K562 erythroleukemia cells. Here, we demonstrate that a stress signal for HSF2 activation occurs when the ubiquitin-proteasome pathway is inhibited. HSF2 DNA-binding activity is induced upon exposure of mammalian cells to the proteasome inhibitors hemin, MG132, and lactacystin, and in the mouse ts85 cell line, which carries a temperature sensitivity mutation in the ubiquitin-activating enzyme (E1) upon shift to the nonpermissive temperature. HSF2 is labile, and its activation requires both continued protein synthesis and reduced degradation. The downstream effect of HSF2 activation by proteasome inhibitors is the induction of the same set of heat shock genes that are induced during heat shock by HSF1, thus revealing that HSF2 affords the cell with a novel heat shock gene-regulatory mechanism to respond to changes in the protein-degradative machinery.
Control of muscle contractile force through indirect high-frequency stimulation. (1983)

With a view to possible application in an orthotic device, study was made of the attenuation of background, indirectly induced muscle tetanus produced by superimposing high-frequency electrical stimulation of the muscle nerve. The effects of varying duration, current and frequency of pulses of the stimulus were outlined, and it was concluded that optimal application might be obtained using a stimulus frequency of 500 Hz, a pulse duration of 50 musec, and varying the frequency to gradate the effect.
Coupling Fluid-Flow and Geomechanics in Dual-Porosity Modeling of Naturally Fractured Reservoirs (1997)

Abstract The purpose of this study is to formulate a coupled fluid-flow/geomechanics model of a naturally fractured reservoir. Fluid flow is modeled within the context of dual-porosity (more generally, overlapping-continuum) concept while geomechanics is modeled following Biot's isothermal, linear poroelastic theory. The development follows along the line of the conventional and existing porous fluid-flow modeling. The commonly used systematic fluid-flow modeling is therefore preserved. We show how the conventional fluid-flow dual-porosity formulations are extended to a coupled fluid-flow/geomechanics model. Interpretation of the pore volumetric changes of the dual continua, fractures and matrix-blocks, and the associated effective stress laws are the most difficult and critical coupling considerations. New relations describing the dual rock volumetric changes are presented. These relations allow a smooth and consistent transition between single-porosity and dual-porosity concepts and are in terms of measurable quantities. Reduction to the single-porosity is presented to demonstrate the conceptual consistency of the proposed model.
Bcr-Abl Resistance Screening Predicts a Limited Spectrum of Point Mutations To Be Associated with Clinical Resistance to the Abl Kinase Inhibitor AMN107. (2005)

Abstract In advanced-phase CML, resistance to imatinib mesylate is frequently associated with point mutations in the Bcr-Abl kinase domain. New, highly potent Abl kinase inhibitors such as AMN107 and BMS-354824, have recently entered clinical trials. Data from analyses of resistant patients will be available not before a large number of resistant patients will have been treated within clinical trials. Therefore, it will be important to generate specific resistance profiles for each compound prior to its therapeutic application. Using a cell-based screening method for resistance of Bcr-Abl positive leukemia to Abl kinase inhibitors, we generated a resistance profile for AMN107 and compared it to the resistance profile of imatinib mesylate. In contrast to imatinib, resistance to AMN107 was associated with a very limited spectrum of Bcr-Abl kinase mutations. While 26 exchanges at 21 positions occured with imatinib, the AMN107 screen revealed eight different exchanges at seven amino acid positions, with four exchanges affecting the P-loop. Novel mutations which have never been observed with imatinib, either in vitro or in resistant patients, emerged in the presence of AMN107 including an F359 exchange to isoleucine and a Q252H/S349L double mutant. In contrast to imatinib, the frequency of resistant colonies dramatically decreased with increasing AMN107 concentrations. Rarely emerging resistant colonies at a concentration of 400 nM AMN107 exclusively contained T315I. With the exception of T315I, all mutations that were identified were effectively suppressed when AMN107 was increased to 2000 nM, a concentration which is achieved in plasma in treated patients. Thus, in this system, increasing the AMN107 concentration to 400 nM prevented the emergence of resistant colonies, with the exception of T315I. Our findings suggest that AMN107 might be superior to imatinib in terms of the development of resistance. Also, AMN107 at clinically relevant concentrations may overcome imatinib resistant disease, including cases with expression of P-loop mutations. However, our study indicates that clinical resistance to AMN107 may be associated with the predominant emergence of T315I. Using this or similar approaches, it will be possible to provide information that translates into combinatorial and sequential treatment strategies and to determine critical plasma concentrations for mutations that might occur during treatment.
Molecular “πPulse” for Total Inversion of Electronic State Population (1998)

Theoretical analysis leads us to the intriguing conclusion that nearly complete electronic population inversion of molecules can be achieved with intense positively chirped broadband laser pulses, as a combined result of vibrational coherence and adiabatic inversion. Strong field quantum calculations demonstrate inversion probabilities of up to 99%. The results are robust with respect to changes in light field parameters as well as to thermal and condensed phase conditions, are supported by experimental evidence, and have many potential applications in chemistry, biochemistry, biology, and physics.
Incorporation of a Non-Nucleotide Bridge into Hairpin Oligonucleotides Capable of High-Affinity Binding to the Rev Protein of HIV-1 (1996)

A bridge containing a rigid trans-stilbene group, -P(O)(O-)O(CH2)3NHC(O)- C6H4-CH=CHC6H4C(O)NH(CH2)3OP(O)(O-)-, has been incorporated into several oligonucleotide sequences based on the minimal Rev Binding Element (RBE) of HIV-1. This bridge was found to be effective as a UUCG tetraloop in stabilizing short RNA duplex structures containing mismatched bases and bulged out nucleotide residues and to be more effective than either a TTTT loop or a triethyleneglycol linker in stabilizing similar DNA structures. Evaluation of stilbene-containing RNA RBE sequences of varying length for their ability to bind the Rev protein of HIV-1 showed that a 22-nucleotide stilbenedicarboxamide conjugate bound Rev almost as well as a 94-base fragment of the Rev Responsive Element (RRE). A DNA hairpin mimetic with the same sequence was incapable of Rev binding. Taken together, these experiments serve as an example for how in vitro selection and chemical modification can be combined to generate high-affinity mimetics of nucleic acid sequence and structure.
TowardsArabidopsisgenome analysis: monitoring expression profiles of 1400 genes using cDNA microarrays (1998)

cDNA microarrays containing 1443 Arabidopsis thaliana genes were analyzed for expression profiles in major organs of Arabidopsis plants. Novel expression profiles were identified for many coding sequences with putative gene identifications. Expression patterns of novel sequences provided clues to their possible functions. The results demonstrate how microarrays containing a large number of Arabidopsis genes can provide a powerful tool for plant gene discovery, functional analysis and elucidation of genetic regulatory networks.