gebhart/patrepeval huggingface dataset:
Measuring mitochondrial respiration in intact single muscle fibers (2011)

Measurement of mitochondrial function in skeletal muscle is a vital tool for understanding regulation of cellular bioenergetics. Currently, a number of different experimental approaches are employed to quantify mitochondrial function, with each involving either mechanically or chemically induced disruption of cellular membranes. Here, we describe a novel approach that allows for the quantification of substrate-induced mitochondria-driven oxygen consumption in intact single skeletal muscle fibers isolated from adult mice. Specifically, we isolated intact muscle fibers from the flexor digitorum brevis muscle and placed the fibers in culture conditions overnight. We then quantified oxygen consumption rates using a highly sensitive microplate format. Peak oxygen consumption rates were significantly increased by 3.4-fold and 2.9-fold by simultaneous stimulation with the uncoupling agent, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and/or pyruvate or palmitate exposure, respectively. However, when calculating the total oxygen consumed over the entire treatment, palmitate exposure resulted in significantly more oxygen consumption compared with pyruvate. Further, as proof of principle for the procedure, we isolated fibers from the mdx mouse model, which has known mitochondrial deficits. We found significant reductions in initial and peak oxygen consumption of 51% and 61% compared with fibers isolated from the wild-type (WT) animals, respectively. In addition, we determined that fibers isolated from mdx mice exhibited less total oxygen consumption in response to the FCCP + pyruvate stimulation compared with the WT mice. This novel approach allows the user to make mitochondria-specific measures in a nondisrupted muscle fiber that has been isolated from a whole muscle.
Facile Recoding of Selenocysteine in Nature (2016)

Selenocysteine (Sec or U) is encoded by UGA, a stop codon reassigned by a Sec-specific elongation factor and a distinctive RNA structure. To discover possible code variations in extant organisms we analyzed 6.4 trillion base pairs of metagenomic sequences and 24 903 microbial genomes for tRNA(Sec) species. As expected, UGA is the predominant Sec codon in use. We also found tRNA(Sec) species that recognize the stop codons UAG and UAA, and ten sense codons. Selenoprotein synthesis programmed by UAG in Geodermatophilus and Blastococcus, and by the Cys codon UGU in Aeromonas salmonicida was confirmed by metabolic labeling with (75) Se or mass spectrometry. Other tRNA(Sec) species with different anticodons enabled E. coli to synthesize active formate dehydrogenase H, a selenoenzyme. This illustrates the ease by which the genetic code may evolve new coding schemes, possibly aiding organisms to adapt to changing environments, and show the genetic code is much more flexible than previously thought.
Relationship between muscle fiber types and sizes and muscle architectural properties in the mouse hindlimb (1994)

Skeletal muscle fiber and architectural properties both contribute to the functional behavior of a muscle. This study uses discriminant analysis and mathematical modeling to identify the structurally and functionally significant properties. The architectural properties of fiber length, muscle length, and pennation angle are found to be the most structurally significant parameters, whereas fiber length, muscle length, and fiber type distribution are found to be most functionally determining. Architectural speed and fiber type do not appear to be complimentary (i.e., the architectural determinant of speed, fiber length, is not associated with fibers of high intrinsic velocity). However, there does seem to be a synergistic relation between the two property classes and force production. Muscles with large physiological cross sectional areas (PCSAs) tend to contain a greater proportion of larger, faster fibers. Structurally or morphologically significant parameters are not always found to have a large functional effect. Pennation angle, though one of the most structurally significant variables, was found to have very little functional effect.
GEPASI: a software package for modelling the dynamics, steady states and control of biochemical and other systems (1993)

GEPASI is a software system for modelling chemical and biochemical reaction networks on computers running Microsoft Windows. For any system of up to 45 metabolites and 45 reactions, each with any user-defined or one of 35 predefined rate equations, one can produce trajectories of the metabolite concentrations and obtain a steady state (if it does exist). When steady-state solutions are produced, elasticity and control coefficients, as defined in metabolic control analysis, are calculated. GEPASI also allows the automatic generation of a sequence of simulations with different combinations of parameter values, effectively scanning a hyper-solid in parameter space. Together with the ability to produce user-defined columnar data files, these features allow for both very quick and systematic study of biochemical pathway models. The source code (in C) is available on request from the author, and while the user interface is dependent on having MS-Windows as the operating system, the numerical part is portable to other operating systems. GEPASI is suitable both for research and educational purposes. Although GEPASI was written with biochemical pathways in mind, it can equally be used to stimulate other dynamical systems.
Peptide-Functionalized Click Hydrogels with Independently Tunable Mechanics and Chemical Functionality for 3D Cell Culture (2010)

Multifunctionalized macromers react via a copper-free click chemistry to form an idealized 3D hydrogel. Subsequently, thiol-containing biomolecules are spatially patterned within the material with precise control over the amount and location of functionalization. Both the network formation and subsequent patterning reactions are fully cytocompatible, allowing these systems to be used to study individual cell behavior at user-defined locations throughout the material.
Adjunctive Immunotherapy of Mycobacterial Infections (2004)

In order to cope with the worldwide increase in the prevalence of multidrug-resistant tuberculosis and Mycobacterium avium complex (MAC) infections, a number of new antimycobacterial drugs have been or are being synthesized and developed. Development of new protocols for chemotherapy of refractory mycobacterioses is also sharing promise. In this context, one promising strategy is to devise regimens to treat patients with refractory mycobacterioses using ordinary antimycobacterial agents in combination with appropriate immunomodulators. This article deals with the following matters: an outline of the host immune response to mycobacterial pathogens, particularly in terms of mobilization of the cytokine network in response to mycobacterial infection, and adjunctive immunotherapy using (1) recombinant immunomodulating cytokines, (especially Th-1 and Th-1-like cytokines such as IFN-gamma, IL-2, IL-12, IL-18 and GM-CSF), (2) inhibitors of immunosuppressive cytokines (TGF-beta) and some proinflammatory tissue-damaging cytokines (TNF-alpha), and (3) immunomodulatory agents such as ATP and its analogs, imidazoquinoline, diethyldithiocarbamate, poloxamer, dibenzopyran, galactosylceramide, nonsteroidal anti-inflammatory drugs, Chinese traditional medicines, levamisole, synthesized mycobacterial oligoDNA, DNA vaccine expressing mycobacterial HSP65 or IL-12, and heat-killed Mycobacterium vaccae. Although adjunctive immunotherapy is fairly efficacious in treating intractable mycobacterioses, it still features serious problems and dilemmas, such as high cost, occasionally severe side effects, and, in many cases, only modest efficacy in potentiating host defense mechanisms against mycobacterial infections, primarily because of the induction of macrophage-deactivating cytokines during the course of long-term administration of adjunctive agents.
Purification and some characteristics of human T-cell growth factor from phytohemagglutinin-stimulated lymphocyte-conditioned media. (1980)

Human T-cell growth factor (TCGF), a mitogenic protein that appears in the media of cultured lymphocytes after phytohemagglutinin-stimulation, has been purified more than 400-fold from serum-free conditioned media by using a sequence of ion exchange chromatography and gel filtration. The purified growth factor elutes as a broad peak from DEAE-Sepharose, focuses diffusely at a pH of about 6.8 on isoelectric focusing (suggesting heterogeneity in electrical charge), has an estimated molecular weight of approximately 23,000 as judged by gel filtration (12,000-13,000 on Na-DodSO4/polyacrylamide gel electrophoresis), is resistant to DNase and RNase, is degraded by trypsin, and does not adhere to any of several lectin-Sepharoses. These characteristics indicate that it is nonglycosylated and protein in nature. The activity of the factor determined by cell counts or [3H]thymidine incorporation in human T lymphoblasts, is stable at room temperature in crude conditioned media, but the partially purified factor requires the addition of albumin or polyethylene glycol to maintain stability. Unlike the crude conditioned media, the purified factor lacks colony-stimulating activity and, unlike lectins, antigens, and crude conditioned media, it does not initiate blastogenesis in peripheral blood lymphocytes but is a selective mitogen for T cells that have undergone blast transformation secondary to exposure to a lectin or antigen. This indicates that the factor is a second signal in the T-cell immune response. The partially purified factor has been used to selectively grow several human T-cell lines, including cells that are cytotoxic to a variety of target cells.
Mortality Associated with Nosocomial Bacteremia Due to Methicillin-Resistant Staphylococcus aureus (1995)

We prospectively studied all cases of Staphylococcus aureus bacteremia that occurred during an extensive outbreak of methicillin-resistant S. aureus (MRSA) in our hospital over a 4-year period (January 1990 through September 1993). We report the results of a comparative analysis of the clinical characteristics and mortality rates among patients with nosocomial bacteremia caused by MRSA (84 cases) or methicillin-susceptible S. aureus (MSSA; 100 cases). The patients with MRSA bacteremia were older than those with MSSA bacteremia (69 years vs. 54 years, respectively; P < .01) and were more likely than those with MSSA bacteremia to have the following predisposing factors: a prolonged hospitalization (32 days vs. 14 days, respectively; P < .01); prior antimicrobial therapy (61% vs. 34%, respectively; P < .01); urinary catheterization (58% vs. 27%, respectively; P < .01); nasogastric tube placement (31% vs. 13%, respectively; P < .01); and prior surgery (45% vs. 31%, respectively; P = .05). Multivariate analysis with use of the stepwise logistic regression method showed a relationship between mortality and the following variables: methicillin resistance (odds ratio [OR], 3), meningitis (OR, 13), and inadequate treatment (OR, 11).
A Comparison of Murine Smooth Muscle Cells Generated from Embryonic versus Induced Pluripotent Stem Cells (2008)

Smooth muscle cell (SMC) differentiation and dedifferentiation play a critical role in the pathogenesis of cardiovascular diseases. The lack of a good and simple in vitro SMC differentiation system has hampered the progress of SMC field for years. The generation of such an in vitro system would be invaluable for exploring molecular mechanisms of SMC differentiation and dedifferentiation. Recently, the establishment of induced pluripotent stem (iPS) cells has offered a novel therapeutic strategy to generate patient-specific stem cell lines. Here we have investigated whether iPS cells are able to differentiate into SMCs in vitro. Mouse iPS cell (O9 and TT025) monolayers were treated with 10(-5) mol/L all-trans retinoid acid (RA). After 8 days of RA treatment, we found that >40% of the O9 iPS cells expressed the SMC-markers including SMalpha-actin and SM myosin heavy chain. Also, we documented that iPS-derived SMCs acquired SMC functional characteristics including contraction and calcium influx in response to stimuli. Moreover, our results indicated that there were differences in SMC-specific gene expression patterns between SMCs derived from O9 and TT025 iPS as well as normal embryonic stem cells. These differences might be due to disparity in the current iPS technology. Taken together, our data have established a simple iPS-SMC system to generate SMCs in vitro, which has tremendous potential to generate individualized SMCs for vascular tissue engineering and personalized drug screening.
Mechanisms of Factor Xa-catalyzed Cleavage of the Factor VIIIa A1 Subunit Resulting in Cofactor Inactivation (2003)

Activation of factor VIII by factor Xa is followed by proteolytic inactivation resulting from cleavage within the A1 subunit (residues 1-372) of factor VIIIa. Factor Xa attacks two sites in A1, Arg(336), which precedes the highly acidic C-terminal region, and a recently identified site at Lys(36). By using isolated A1 subunit as substrate for proteolysis, production of the terminal fragment, A1(37-336), was shown to proceed via two pathways identified by the intermediates A1(1-336) and A1(37-372) and generated by initial cleavage at Arg(336) and Lys(36), respectively. Appearance of the terminal product by the former pathway was 7-8-fold slower than the product obtained by the latter pathway. The isolated A1 subunit was cleaved slowly, independent of the presence of phospholipid. The A1/A3-C1-C2 dimer demonstrated an approximately 3-fold increased cleavage rate constant, and inclusion of phospholipid further enhanced this value by approximately 2-fold. Although association of A1 or A1(37-372) with A3-C1-C2 enhanced the rate of cleavage at Arg(336), inclusion of A3-C1-C2 did not affect the cleavage at Lys(36) in A1(1-336). A synthetic peptide 337-372 blocked the cleavage at Lys(36) (IC(50) = 230 microm) while showing little if any effect on cleavage at Arg(336). Proteolysis at Lys(36), and to a lesser extent Arg(336), was inhibited in a dose-dependent manner by heparin. These results suggest that inactivating cleavages catalyzed by factor Xa at Lys(36) and Arg(336) are regulated in part by the A3-C1-C2 subunit. Furthermore, cleavage at Lys(36) appears to be selectively modulated by the C-terminal acidic region of A1, a region that may interact with factor Xa via its heparin-binding exosite.
The Protein Kinase Complement of the Human Genome (2002)

We have catalogued the protein kinase complement of the human genome (the "kinome") using public and proprietary genomic, complementary DNA, and expressed sequence tag (EST) sequences. This provides a starting point for comprehensive analysis of protein phosphorylation in normal and disease states, as well as a detailed view of the current state of human genome analysis through a focus on one large gene family. We identify 518 putative protein kinase genes, of which 71 have not previously been reported or described as kinases, and we extend or correct the protein sequences of 56 more kinases. New genes include members of well-studied families as well as previously unidentified families, some of which are conserved in model organisms. Classification and comparison with model organism kinomes identified orthologous groups and highlighted expansions specific to human and other lineages. We also identified 106 protein kinase pseudogenes. Chromosomal mapping revealed several small clusters of kinase genes and revealed that 244 kinases map to disease loci or cancer amplicons.
Regio‐ and Stereoselective Reduction of Diketones and Oxidation of Diols by Biocatalytic Hydrogen Transfer (2006)

Abstract The asymmetric reduction of symmetrical and nonsymmetrical diketones as well as the stereoselective oxidation of various diols by biocatalytic hydrogen transfer was investigated by employing lyophilized cells of Rhodococcus ruber DSM 44541 containing alcohol dehydrogense ADH‐‘A’. Symmetrical and nonsymmetrical diketones at the (ω‐1)‐ and (ω‐2)‐positions are reduced to the Prelog product with high stereopreference, while sterically more demanding ketone moieties, for example those at the (ω‐3)‐position, remain unchanged. For the oxidation mode, differentiation between primary and secondary alcohols is achieved, and the ( S )‐configured secondary alcohols at the (ω‐1)‐ and (ω‐2)‐positions are oxidized preferentially. (© Wiley‐VCH Verlag GmbH &amp; Co. KGaA, 69451 Weinheim, Germany, 2006)
Libraries for each human chromosome, constructed from sorter-enriched chromosomes by using linker-adaptor PCR. (1993)

We describe here the production of complex libraries enriched in sequences from each human chromosome type, starting with only a few thousand sorter-purified chromosomes. In this procedure, DNA is extracted from the sorted chromosomes, digested to completion by using the frequently cutting restriction endonuclease Sau3A1, and ligated, on each end, to an adaptor oligonucleotide. These fragments are then amplified using PCR with a sequence homologous to the adaptor oligonucleotide as a primer. We have used this procedure to produce PCR libraries for each of the 24 human chromosomes. These libraries were characterized by gel electrophoresis and found to be composed of a continuum of sequences ranging in size from a few hundred to approximately 1,000 bp. The libraries, when used as probes for fluorescence in situ hybridization, stained the target chromosomes more or less continuously, even after PCR amplification for more than 200 cycles. These libraries are useful as hybridization probes to facilitate molecular cytogenetic studies and as sources of probes either for identification of polymorphic short tandemly repeated sequences or for development of sequence-tagged sites.
Staphylococcus aureus endocarditis at a community teaching hospital, 1980 to 1991. An analysis of 106 cases (1994)

<h3>Background:</h3> The clinical diagnosis of infective endocarditis due to<i>Staphylococcus aureus</i>can be difficult, and many patients with this disease are only diagnosed post mortem. There are few published reports of large series of patients with<i>S aureus</i>endocarditis and none from a community hospital. I reviewed the clinical and laboratory findings of a large number of patients with<i>S aureus</i>endocarditis in a community hospital. <h3>Methods:</h3> I reviewed medical records identified through consultation records,<i>International Classification of Diseases, Ninth Edition</i>codes, and autopsy records of patients who fulfilled the criteria for the diagnosis of<i>S aureus</i>endocarditis during 1980 to 1991. <h3>Results:</h3> During the 12-year period, there were 106 cases, for a prevalence of 0.34 per 1000 admissions. Ninety-three (87.7%) of these patients were seen by me. The patients' ages ranged from 12 to 83 years (median, 61 years). Eighteen cases were nosocomial (15 were associated with intravascular catheters). Twenty-one patients were injecting drug users. Severe back pain was the chief complaint in nine patients. Twenty-seven patients had no heart murmur at the time of diagnosis. The overall mortality was 25.5%. <h3>Conclusions:</h3> Age 60 years or older, female gender, community-acquired infection, absence of heart murmur, presence of congestive heart failure, or central nervous system involvement was associated with higher mortality. Tricuspid valve endocarditis alone was associated with lower mortality. (Arch Intern Med. 1994;154:2330-2335)
Characterisation of endogenous A2A and A2B receptor-mediated cyclic AMP responses in HEK 293 cells using the GloSensor™ biosensor: Evidence for an allosteric mechanism of action for the A2B-selective antagonist PSB 603 (2017)

Endogenous adenosine A<sub>2B</sub> receptors (A<sub>2B</sub>AR) mediate cAMP accumulation in HEK 293 cells. Here we have used a biosensor to investigate the mechanism of action of the A<sub>2B</sub>AR antagonist PSB 603 in HEK 293 cells. The A<sub>2A</sub> agonist CGS 21680 elicited a small response in these cells (circa 20% of that obtained with NECA), suggesting that they also contain a small population of A<sub>2A</sub> receptors. The responses to NECA and adenosine were antagonised by PSB 603, but not by the selective A<sub>2A</sub>AR antagonist SCH 58261. In contrast, CGS 21680 responses were not antagonised by high concentrations of PSB 603, but were sensitive to inhibition by SCH 58261. Analysis of the effect of increasing concentrations of PSB 603 on the response to NECA indicated a non-competitive mode of action yielding a marked reduction in the NECA E<sub>MAX</sub> with no significant effect on EC<sub>50</sub> values. Kinetics analysis of the effect of PSB 603 on the A<sub>2B</sub>AR-mediated NECA responses confirmed a saturable effect that was consistent with an allosteric mode of antagonism. The possibility that PSB 603 acts as a negative allosteric modulator of A<sub>2B</sub>AR suggests new approaches to the development of therapeutic agents to treat conditions where adenosine levels are high.
Advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction (2002)

In recent years, lentiviral expression systems have gained an unmatched reputation among the gene therapy community for their ability to deliver therapeutic transgenes into a wide variety of difficult-to-transfect/transduce target tissues (brain, hematopoietic system, liver, lung, retina) without eliciting significant humoral immune responses. We have cloned a construction kit-like self-inactivating lentiviral expression vector family which is compatible to state-of-the-art packaging and pseudotyping technologies and contains, besides essential cis-acting lentiviral sequences, (i) unparalleled polylinkers with up to 29 unique sites for restriction endonucleases, many of which recognize 8 bp motifs, (ii) strong promoters derived from the human cytomegalovirus immediate-early promoter (P(hCMV)) or the human elongation factor 1alpha (P(hEF1)(alpha)), (iii) P(hCMV-) or P(PGK-) (phosphoglycerate kinase promoter) driven G418 resistance markers or fluorescent protein-based expression tracers and (iv) tricistronic expression cassettes for coordinated expression of up to three transgenes. In addition, we have designed a size-optimized series of highly modular lentiviral expression vectors (pLenti Module) which contain, besides the extensive central polylinker, unique restriction sites flanking any of the 5'U3, R-U5-psi+-SD, cPPT-RRE-SA and 3'LTR(DeltaU3) modules or placed within the 5'U3 (-78 bp) and 3'LTR(DeltaU3) (8666 bp). pLentiModule enables straightforward cassette-type module swapping between lentiviral expression vector family members and facilitates the design of Tat-independent (replacement of 5'LTR by heterologous promoter elements), regulated and self-excisable proviruses (insertion of responsive operators or LoxP in the 3'LTR(DeltaU3) element). We have validated our lentiviral expression vectors by transduction of a variety of insect, chicken, murine and human cell lines as well as adult rat cardiomyocytes, rat hippocampal slices and chicken embryos. The novel multi-purpose construction kit-like vector series described here is compatible with itself as well as many other (non-viral) mammalian expression vectors for straightforward exchange of key components (e.g. promoters, LTRs, resistance genes) and will assist the gene therapy and tissue engineering communities in developing lentiviral expression vectors tailored for optimal treatment of prominent human diseases.