gebhart/patrepeval huggingface dataset:
Construction and Characterization of Mutants of the TEM-1 β-Lactamase Containing Amino Acid Substitutions Associated with both Extended-Spectrum Resistance and Resistance to β-Lactamase Inhibitors (1999)

Extended-spectrum TEM beta-lactamases (ESBLs) do not usually confer resistance to beta-lactamase inhibitors such as clavulanate or tazobactam. To investigate the compatibility of the two phenotypes we used site-directed mutagenesis of the bla(TEM-1) gene to introduce into the TEM-1 beta-lactamase amino acid substitutions that confer the ESBL phenotype: TEM-12 (Arg164-->Ser), TEM-26 (Arg164-->Ser plus Glu104-->Lys), TEM-19 (Gly238-->Ser), and TEM-15 (Gly238-->Ser plus Glu104-->Lys). These were combined with three sets of substitutions that confer inhibitor resistance: TEM-31 (Arg244-->Cys), TEM-33 (Met69-->Leu), and TEM-35 (Met69-->Leu and Asn276-->Asp). Introduction of the Arg244-->Cys substitution gave rise to inhibitor-resistant hybrid enzymes that either lost ESBL activity (TEM-12, TEM-15, and TEM-19) or had reduced activity (TEM-26) against ceftazidime. In contrast, the introduction of Met69-->Leu or Met69-->Leu plus Asn276-->Asp substitutions did not significantly affect the abilities of the enzymes to confer resistance to ceftazidime, although increased susceptibility to cefotaxime was observed with Escherichia coli strains that expressed the TEM-19 and TEM-26 beta-lactamases. With the exception of the TEM-12 beta-lactamase, introduction of the Met69-->Leu substitution did not give rise to enzymes with increased resistance to clavulanate compared to that of the TEM-1 beta-lactamase. However, introduction of the double substitution Met69-->Leu plus Asn276-->Asp in the ESBLs did give rise to low-level (TEM-19, TEM-15, and TEM-26) or moderate-level (TEM-12) clavulanate resistance. None of the hybrid enzymes were as resistant to clavulanate as the corresponding inhibitor-resistant TEM beta-lactamase mutant, suggesting that active-site configuration in the ESBLs limits the degree of clavulanate resistance conferred.
Screening for mutations by expressing patient cDNA segments inE. coli: Homocystinuria due to cystathionine β-synthase deficiency (1992)

Deficiency of cystathionine beta-synthase (CBS) causes the most common form of inherited homocystinuria. We developed a simple CBS expression system in E. coli to screen for pathogenic mutations in affected individuals. Portions of patient cDNAs were amplified by PCR and used to replace the corresponding segments of normal human CBS cDNA in the bacterial expression plasmid pHCS3. Hybrid CBS was expressed in E. coli and the segments of patient's cDNA which extinguished CBS activity were sequenced to identify the mutation. The first study of a pyridoxine-responsive patient using this screen revealed that of the clones which contained either the middle or the 3'-portion of his cDNA, about half were devoid of catalytic activity. Subsequent sequencing of the affected segments confirmed a compound heterozygosity for a maternal T833-->C transition (I278T) and for a paternal A-->C transversion in the intron 11 splice acceptor. The latter mutation leads to an in-frame deletion of exon 12 (nt 1224-1358, amino acids W408 to G453). This bacterial expression system proved to be a rapid screening method for localizing pathogenic mutations in CBS, allowing us to sequence the affected portions of mutant cDNA within 7-10 days of harvesting cultured fibroblasts.
The possible use of HLA-G1 and G3 in the inhibition of NK cell-mediated swine endothelial cell lysis (2001)

The splicing isoform of HLA-G that is expressed in xenogeneic cells, and its effect on NK-mediated direct cytotoxicity was examined, using stable Chinese hamster ovary (CHO) cell or swine endothelial cell (SEC) transfectants. cDNAs of HLA-G (G1 and G3) and human beta2-microglobulin were prepared and subcloned into the expression vector, pCXN. The transfected HLA-G1 was easily expressed on SEC, and co-transfection with human beta2-microglobulin led to an enhanced level of HLA-G1 expression, as evidenced by flow cytometry. The expressed HLA-G1 significantly suppressed NK-mediated SEC cell lysis, which is an in vitro delayed-type rejection model of a xenograft. On the other hand, the swine leucocyte antigen (SLA) class I molecules could be up-regulated as the result of the transfection of human beta2-microglobulin, but did not down-regulate human NK-mediated SEC lysis. The HLA-G3 was not expressed on CHO and SEC in contrast to HLA-G1, as the result of the transfection. The gene introduction of HLA-G3 in SEC showed no protective effect from human NK cells. However, indirect evidence demonstrated that HLA-G3 transfection resulted in HLA-E expression, but not itself, when transfected to the human cell line, 721.221, thus providing some insight into its natural function in human cells. The present findings suggest that the expression of HLA-G1 on the cell surface could serve as a new approach to overcoming NK-mediated immunity to xenografts.
Thiamine metabolism in the liver of mice with Ehrlich ascites carcinoma. (1982)

Thiamine pyrophosphate (TPP) content, activities of thiamine pyrophosphokinase (TPKase), thiamine pyrophosphatase, transketolase (TK), pyruvate (PDG) and oxoglutarate dehydrogenases (OGDG) were measured in the liver and cells of Ehrlich ascites carcinoma (EAC) on the 5th, 10th and 15th day after transplantation of the tumor to mice fed a thiamine-deficient diet. The TPP level gradually decreased in the liver of tumor-bearing mice but remained constant in tumor cells (1.06 +/- 0.02 microgram/g tissue). Deprivation of dietary thiamine lowered the liver TPP level even to a greater extent, and subsequent daily 10 micrograms thiamine/mouse injections did not restore it. The TPKase activity in the liver of mice with EAC decreased by 24% and in thiamine deficiency, by 44%. The liver PDG, OGDG and TK activities were reduced but slightly in mice with EAC, whereas thiamine deprivation resulted in a decrease of the enzyme activities: PDG by 60%, OGDG by 25% and TK by 45%. TK activity in tumor cells was 90 mumol S-7-P/g tissue/h, and the TPP effect amounted to 24%. Thiamine deprivation decreased the TK activity by 45% and raised the TPP effect up to 180%. Thiamine injections restored the TK activity in tumor cells and reduced the TPP effect.
Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma (2008)

Prenatal diagnosis of monogenic diseases, such as cystic fibrosis and β-thalassemia, is currently offered as part of public health programs. However, current methods based on chorionic villus sampling and amniocentesis for obtaining fetal genetic material pose a risk to the fetus. Since the discovery of cell-free fetal DNA in maternal plasma, the noninvasive prenatal assessment of paternally inherited traits or mutations has been achieved. Due to the presence of background maternal DNA, which interferes with the analysis of fetal DNA in maternal plasma, noninvasive prenatal diagnosis of maternally inherited mutations has not been possible. Here we describe a digital relative mutation dosage (RMD) approach that determines if the dosages of the mutant and wild-type alleles of a disease-causing gene are balanced or unbalanced in maternal plasma. When applied to the testing of women heterozygous for the CD41/42 (–CTTT) and hemoglobin E mutations on HBB , digital RMD allows the fetal genotype to be deduced. The diagnostic performance of digital RMD is dependent on interplay between the fractional fetal DNA concentration and number of DNA molecules in maternal plasma. To achieve fetal genotype diagnosis at lower volumes of maternal plasma, fetal DNA enrichment is desired. We thus developed a digital nucleic acid size selection (NASS) strategy that effectively enriches the fetal DNA without additional plasma sampling or experimental time. We show that digital NASS can work in concert with digital RMD to increase the proportion of cases with classifiable fetal genotypes and to bring noninvasive prenatal diagnosis of monogenic diseases closer to reality.
[Chronic pelvic pain treatment with posterior tibial nerve stimulation]. (2011)

In conclusion, the use of PTNS in the treatment of chronic pelvic pain shows encouraging results in patients not responding to standard analgesic therapy.
Step-and-Repeat Photo-Nanoimprint System Using Active Orientation Head (2004)

An active orientation head was designed and installed into a step-and-repeat photo-nanoimprint system. The characteristics of the passive part of the active orientation head and compliant contact function as a basic function were evaluated. Feedback control of the active orientation head resulted in 50% load deviation at four locations to within 10%. In addition, asymmetrical load control was realized, which is one of the additional functions of the active orientation head. The automatic photo-nanoimprint sequence was demonstrated, in which the imprint load was controlled to within 1.3% of the target value. In 50 min operation, 25 dies were imprinted on a 4-inch wafer. Bright spots are visible to the eye in the dark-field illumination at each location where patterns should be found. Similar patterns 100–300 nm wide on different dies were observable by optical microscopy. Scanning electron microscope revealed smooth patterns with a half pitch of 100 nm fabricated by the step-and-repeat photo-nanoimprint system.
famRCA-RACE: A ROLLING CIRCLE AMPLIFICATION RACE FOR ISOLATING A FAMILY OF HOMOLOGOUS cDNAs IN ONE REACTION AND ITS APPLICATION TO OBTAIN NAC GENES TRANSCRIPTION FACTORS FROM CROCUS (CROCUS SATIVUS) FLOWER (2010)

We describe an improvement of the RCA-RACE (rolling circle amplification-rapid amplification of cDNA ends) method, called family RCA-RACE (famRCA-RACE). The method is based on the generation of circular cDNA fragments, followed by rolling circle amplification of the circular cDNA using phi29 DNA polymerase and the application of PCR using degenerate outworking primers, designed for a conserved region of homologous genes, that allows the isolation of homologous cDNA sequences expressed in the mRNA preparation in a single polymerase chain reaction (PCR). As an example we present the isolation of seven NAC-like transcription factors cDNA sequences expressed in Crocus sativus flower, used for saffron production. Sequence alignment revealed that CsatNAC proteins contain the typical domain structure of plant NAC proteins, consisting of the conserved N-terminal NAC domain used to design the primers and the five subdomains. Phylogenetic analysis revealed that CsatNAC proteins fall in subgroup I of the NAC family of proteins.
Different functional roles of T1R subunits in the heteromeric taste receptors (2004)

The T1R receptors, a family of taste-specific class C G protein-coupled receptors, mediate mammalian sweet and umami tastes. The structure-function relationships of T1R receptors remain largely unknown. In this study, we demonstrate the different functional roles of T1R extracellular and transmembrane domains in ligand recognition and G protein coupling. Similar to other family C G protein-coupled receptors, the N-terminal Venus flytrap domain of T1R2 is required for recognizing sweeteners, such as aspartame and neotame. The G protein coupling requires the transmembrane domain of T1R2. Surprisingly, the C-terminal transmembrane domain of T1R3 is required for recognizing sweetener cyclamate and sweet taste inhibitor lactisole. Because T1R3 is the common subunit in the sweet taste receptor and the umami taste receptor, we tested the interaction of lactisole and cyclamate with the umami taste receptor. Lactisole inhibits the activity of the human T1R1/T1R3 receptor, and, as predicted, blocked the umami taste of l-glutamate in human taste tests. Cyclamate does not activate the T1R1/T1R3 receptor by itself, but potentiates the receptor's response to l-glutamate. Taken together, these findings demonstrate the different functional roles of T1R3 and T1R2 and the presence of multiple ligand binding sites on the sweet taste receptor.
Here come the septins: novel polymers that coordinate intracellular functions and organization (2005)

Septins are conserved GTP-binding proteins that associate with cellular membranes and the actin and microtubule cytoskeletons. They polymerize to form filamentous structures that act as diffusion barriers between different membrane domains and as molecular scaffolds for membrane- and cytoskeleton-binding proteins. In yeast, septins are central to the spatio-temporal coordination of membrane polarity and cell division, but the roles of their mammalian counterparts have remained poorly understood. However, recent findings have shed light on the dynamics and regulation of mammalian septin assembly and our understanding of septin functions in cytoskeleton and membrane organization. The mammalian septins appear to form a novel network of hetero-polymers that are multi-functional, inter-changeable and respond dynamically to signals that coordinate events at the interface between cytoskeleton and membrane biology. Hence, studies of these molecules might provide new insights not only into how cells coordinate their functions, but also into the pathogenesis of cancer and other diseases in which septins are abnormally expressed.
Hyperosmotic Stress Stimulates Promoter Activity and Regulates Cellular Utilization of the Serum- and Glucocorticoid-inducible Protein Kinase (Sgk) by a p38 MAPK-dependent Pathway (2000)

We have established that the serum- and glucocorticoid-inducible protein kinase (Sgk) is a new component of the hyperosmotic stress response. Treatment of NMuMg mammary epithelial cells with the organic osmolyte, sorbitol, caused the stable accumulation of Sgk transcripts and protein after an approximately 4-h lag. Transient transfection of a series of sgk-CAT reporter plasmids containing either 5' deletions or continuous 6-base pair substitutions identified a hyperosmotic stress-regulated element that is GC-rich and is necessary for the sorbitol stimulation of sgk gene promoter activity. Gel shift analysis identified four major DNA-protein complexes in the hyperosmotic stress-regulated element that, by competition with excess consensus wild type and mutant oligonucleotides and by antibody supershifts, contains the Sp1 transcription factor. Several lines of evidence suggest that the p38 MAPK signaling pathway mediates the hyperosmotic stress stimulation of sgk gene expression. Treatment with pharmacological inhibitors of p38 MAPK or with a dominant negative form of MKK3, an upstream regulator of p38 MAPK, significantly reduced or ablated the sorbitol induction of sgk promoter activity or protein production. Using an in vitro peptide transphosphorylation assay, sorbitol treatment activates either endogenous or exogenous Sgk that is localized to the cytoplasmic compartment. Thus, we propose that the stimulated expression of enzymatically active Sgk after sorbitol treatment is a newly defined component of the p38 MAPK-mediated response to hyperosmotic stress.
Performance Enhancement for Network I/O Virtualization with Efficient Interrupt Coalescing and Virtual Receive-Side Scaling (2012)

Virtualization is a key technology in cloud computing; it can accommodate numerous guest VMs to provide transparent services, such as live migration, high availability, and rapid checkpointing. Cloud computing using virtualization allows workloads to be deployed and scaled quickly through the rapid provisioning of virtual machines on physical machines. However, I/O virtualization, particularly for networking, suffers from significant performance degradation in the presence of high-speed networking connections. In this paper, we first analyze performance challenges in network I/O virtualization and identify two problems-conventional network I/O virtualization suffers from excessive virtual interrupts to guest VMs, and the back-end driver does not efficiently use the computing resources of underlying multicore processors. To address these challenges, we propose optimization methods for enhancing the networking performance: 1) Efficient interrupt coalescing for network I/O virtualization and 2) virtual receive-side scaling to effectively leverage multicore processors. These methods are implemented and evaluated with extensive performance tests on a Xen virtualization platform. Our experimental results confirm that the proposed optimizations can significantly improve network I/O virtualization performance and effectively solve the performance challenges.
PDE Inhibition and cognition enhancement (2012)

There has been an increasing interest in the development of phosphodiesterase (PDE) inhibitors for the treatment of cognitive dysfunctions. In this editorial, the mechanism of action of PDEs is briefly described, while the effects of different PDE inhibitors in preclinical models are reviewed. Based on the expression of PDE mRNA in the human brain, it is suggested that PDE1 and PDE10 inhibitors are strong candidates for the development of cognition enhancers. However, the complex nature of the expression of PDEs in the brain warrants further research into the role of PDEs in the signaling pathways in brain circuits. The development of PDE inhibitors, which are selective for PDE splicing isoforms, may be promising for future drug development.
Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide (2007)

Sequence-specific interference with the nuclear pre-mRNA splicing machinery has received increased attention as an analytical tool and for development of therapeutics. It requires sequence-specific and high affinity binding of RNaseH-incompetent DNA mimics to pre-mRNA. Peptide nucleic acids (PNA) or phosphoramidate morpholino oligonucleotides (PMO) are particularly suited as steric block oligonucleotides in this respect. However, splicing correction by PNA or PMO conjugated to cell penetrating peptides (CPP), such as Tat or Penetratin, has required high concentrations (5-10 microM) of such conjugates, unless an endosomolytic agent was added to increase escape from endocytic vesicles. We have focused on the modification of existing CPPs to search for peptides able to deliver more efficiently splice correcting PNA or PMO to the nucleus in the absence of endosomolytic agents. We describe here R6-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) as the most active of all CPPs tested so far in a splicing correction assay in which masking of a cryptic splice site allows expression of a luciferase reporter gene. Efficient and sequence-specific correction occurs at 1 muM concentration of the R6Pen-PNA705 conjugate as monitored by luciferase luminescence and by RT-PCR. Some aspects of the R6Pen-PNA705 structure-function relationship have also been evaluated.
Multifunctional Gold Nanoparticle−Peptide Complexes for Nuclear Targeting (2003)

The ability of peptide-modified gold nanoparticles to target the nucleus of HepG2 cells was explored. Five peptide/nanoparticle complexes were investigated, particles modified with (1) the nuclear localization signal (NLS) from the SV 40 virus; (2) the adenovirus NLS; (3) the adenovirus receptor-mediated endocytosis (RME) peptide; (4) one long peptide containing the adenovirus RME and NLS; and (5) the adenovirus RME and NLS peptides attached to the nanoparticle as separate pieces. Gold nanoparticles were used because they are easy to identify using video-enhanced color differential interference contrast microscopy, and they are excellent scaffolds from which to build multifunctional nuclear targeting vectors. For example, particles modified solely with NLS peptides were not able to target the nucleus of HepG2 cells from outside the plasma membrane, because they either could not enter the cell or were trapped in endosomes. The combination of NLS/RME particles (4) and (5) did reach the nucleus; however, nuclear targeting was more efficient when the two signals were attached to nanoparticles as separate short pieces versus one long peptide. These studies highlight the challenges associated with nuclear targeting and the potential advantages of designing multifunctional nanostructured materials as tools for intracellular diagnostics and therapeutic delivery.
A Role of Serum-Based Neuronal and Glial Markers as Potential Predictors for Distinguishing Severity and Related Outcomes in Traumatic Brain Injury (2015)

S100B, GFAP, and UCH-L1 serum level of were significantly increased in TBI according to severity and associated clinical outcomes. Biomarkers have potential utility as diagnostic, prognostic, and therapeutic adjuncts in the setting of TBI.
Quantitation of L-selectin and CD18 expression on rabbit neutrophils during CD18-independent and CD18-dependent emigration in the lung. (1994)

In the systemic circulation, the leukocyte adhesion molecule, L-selectin facilitates the initial adhesion of the neutrophil to the inflamed endothelium, whereas CD11/CD18 is essential to transendothelial migration. Previous work from our laboratory showed that neutrophil emigration in the lung occurs through either a CD18-independent or CD18-dependent mechanism, depending on the inflammatory stimulus. This study quantitated and compared the surface expression of L-selectin and CD18 on neutrophils in the lungs of rabbits during emigration toward Streptococcus pneumoniae (a CD18-independent stimulus) and Escherichia coli endotoxin (a CD18-dependent stimulus). Ultrathin frozen lung tissue sections were immunogold labeled for 1-selectin and CD18, and gold particles were quantitated on intravascular, interstitial, and airspace neutrophils by transmission electron microscopy. The results show that CD18-independent neutrophil emigration was associated with L-selectin down-modulation (78%) and CD18 up-modulation (260%) on intravascular neutrophils, before emigration. A similar alteration in the expression of L-selectin and CD18 was observed during CD18-dependent neutrophil emigration, but only on neutrophils that emigrated into the interstitium and airspace. In emigration induced by either stimulus, alterations in L-selectin and CD18 expression were restricted to the inflammatory focus and emigrated airspace neutrophils consistently expressed greater levels of CD18 than intravascular and interstitial neutrophils. We conclude that before emigration, L-selectin and CD18 expression on intravascular neutrophils is altered only during CD18-independent emigration and only on those neutrophils within the inflammatory focus. The increased CD18 expression on airspace neutrophils may facilitate bacterial phagocytosis.