gebhart/patrepeval huggingface dataset:
Principles of Machine Learning-Guided Protein Engineering (2020)

Protein engineering has enormous academic, industrial, and biomedical potential. However, it is limited by our ability to efficiently explore astronomically large sequence spaces to find rare high-functioning variants. In this thesis, we find that when screening or selection capacity is high, directed evolution is often sufficient to find such variants. In such settings, machine learning can be used to explore distant regions of sequence space that may serve as substrates for directed evolution. However, under resource constraints typical of many high-value protein systems and late-stage or high-fidelity engineering efforts, screening and selection capacity is low, making directed evolution substantially less effective. Toward this end, we developed a semi-supervised machine learning framework, UniRep, that from scratch and from sequence alone learned to distill the fundamental features of a protein – including biophysical, structural, and evolutionary information – into a holistic statistical representation. Trained on a vast, exponentially growing, unlabeled sequence database, UniRep not only enables state-of-the-art predictive performance on a diverse variety of protein informatics tasks, but also when combined with in silico directed evolution, enables engineering in resource constrained settings where only a small number – low-N – of variants can be functionally characterized. Taken together, we conclude that semi- and self-supervised machine learning, process virtualization, and a few carefully chosen experimental measurements may rapidly accelerate and reduce the costs of protein engineering in a manner that other (semi-)rational design approaches and directed evolution cannot.
Signal processing with lapped transforms (1992)

Applications of transforms in signal processing signal processing in subbands lapped orthogonal transforms the modulated lapped transform heirarchical lapped transforms applications of lapped transforms.
Reengineering the catalytic lysine of aspartate aminotransferase by chemical elaboration of a genetically introduced cysteine (1991)

The active-site essential catalytic residue of aspartate aminotransferase, Lys 258, has been converted to Cys (K258C) by site-directed mutagenesis. This mutant retains less than 10(-6) of the wild-type activity with L-aspartate. The deleted general base was functionally replaced by selective (with respect to the other five cysteines in wild type) aminoethylation of the introduced Cys 258 with (2-bromoethyl)amine following reversible protection of the nontarget sulfhydryl groups at different stages of unfolding. The chemically elaborated mutant (K258C-EA) is 10(5) times more reactive than is K258C and has a kcat value of approximately 7% of that of wild type (WT). Km and KI values are similar to those for WT. The acidic pKa controlling V/KAsp is shifted from 7.3 (WT) to 6.0 (mutant). V/K values for amino acids are approximately 3% of those found for WT, whereas they are approximately 20% for keto acids. The value of DV increases from 1.6 for WT to 3.4 for the mutant, indicating that C alpha proton abstraction constitutes a more significant kinetic barrier for the latter enzyme. A smaller, but still significant, increase in D(V/KAsp) from 1.9 in WT to 3.0 in the mutant shows that the forward and reverse commitment factors are inverted by the mutation. The acidic limb of the V/KAsp versus pH profile, is lowered by 1.3 pH units, probably reflecting the similar difference in the basicity of the epsilon-NH2 group in gamma-thialysine versus that in lysine.(ABSTRACT TRUNCATED AT 250 WORDS)
Severe secondary deficiency of von Willebrand factor–cleaving protease (ADAMTS13) in patients with sepsis-induced disseminated intravascular coagulation: its correlation with development of renal failure (2005)

Deficiency of ADAMTS13 is found in patients with thrombotic thrombocytopenic purpura (TTP), and the genetic defects in the ADAMTS13 gene or the autoantibody against ADAMTS13 is thought to be responsible for the development of TTP. The clinical correlation and mechanisms of secondary ADAMTS13 deficiency in other disease states were investigated. In addition to TTP, ADAMTS13 levels were severely decreased in patients with sepsis-induced disseminated intravascular coagulation (DIC). The incidence of acute renal failure and serum creatinine levels in patients with ADAMTS13 activity levels lower than 20% (incidence, 41.2%; creatinine, 160 +/- 150 microM [1.81 +/- 1.70 mg/dL]) (P < .05) were significantly higher than they were in patients with ADAMTS13 activity levels higher than 20% (incidence, 15.4%; creatinine, 84 +/- 67 microM [0.95 +/- 0.76 mg/dL]) (P < .01). Additionally, unusually large von Willebrand factor multimers were detected in 26 (51.0%) of 51 patients with ADAMTS13 activity levels lower than 20%. Lower molecular weight forms of ADAMTS13 were found in the plasma of patients with sepsis-induced DIC, suggesting that the deficiency of ADAMTS13 was partially caused by its cleavage by proteases in addition to decreased synthesis in the liver. These data suggested that severe secondary ADAMTS13 deficiency can be associated with sepsis-induced DIC and may contribute to the development of renal failure.
A comparative analysis of the activity of ligands acting at P2X and P2Y receptor subtypes in models of neuropathic, acute and inflammatory pain (2010)

Our results show that antagonism at P2X1, P2Y12, and P2X7 receptors and agonism at P2Y1 receptors define promising therapeutic strategies in acute, neuropathic, and inflammatory pain respectively.
Selective decrease in cell surface expression and mRNA level of the 55-kDa tumor necrosis factor receptor during differentiation of HL-60 cells into macrophage-like but not granulocyte-like cells (1992)

Expression of the two known receptors for TNF was studied in the promyelocytic leukemia cell line HL-60 before and after differentiation of the cells along the granulocyte lineage (induced by incubation with retinoic acid), or along the macrophage lineage (induced by incubation with the phorbol diester, PMA). The extent of inhibition of TNF binding by receptor-specific antisera, as well as the size of the complexes formed after cross-linking TNF to its receptors on intact cells, indicated that both receptor species were expressed on the surface of the undifferentiated HL60 cells. Differentiation into granulocyte-like cells resulted in some increase in TNF binding. The increase was apparently due to enhanced expression of the 75-kDa TNF-R, whereas the amounts of the 55-kDa TNF-R did not change significantly. In contrast, in HL-60 cells induced to differentiate into macrophage-like cells, expression of the 55-kDa TNF-R species was completely abolished. The pattern of TNF-R expression in the differentiated HL-60 cells was similar to that observed in leukocytes isolated from peripheral blood: on granulocytes, there were about equal amounts of both receptor species, whereas on monocytes the 75-kDa receptor was predominant. The loss of 55-kDa receptors during differentiation of HL-60 cells into macrophage-like cells was accompanied by a pronounced decrease in the level of the mRNA for that receptor, suggesting that at least part of the change in TNF-R expression is due to mechanisms that control the amounts of receptor mRNA. Although little is yet known regarding the functional differences between the two receptor species, marked changes in the pattern of their expression, as observed during HL-60 cell differentiation, are likely to alter the kind of response of the cells to TNF and may therefore play an important role in the coordination of TNF effects in the organism.
Free fatty acids activate a high-affinity saturable pathway for degradation of low-density lipoproteins in fibroblasts from a subject homozygous for familial hypercholesterolemia (1992)

This paper describes a mechanism for degradation of low-density lipoprotein (LDL) in fibroblasts unable to synthesize the LDL receptor. In this cell line, long-chain free fatty acids (FFA) activated 125I-LDL uptake; unsaturated FFA were the most efficient. The first step of this pathway was the binding of LDL apoB to a single class of sites on the plasma membrane and was reversible in the presence of greater than or equal to 10 mM suramin. Binding equilibrium was achieved after a 60-90-min incubation at 37 degrees C with 1 mM oleate; under these conditions, the apparent Kd for 125I-LDL binding was 12.3 micrograms/mL. Both cholesterol-rich (LDL and beta-VLDL) and triglyceride-rich (VLDL) lipoproteins, but not apoE-free HDL, efficiently competed with 125I-LDL for this FFA-induced binding site. After LDL bound to the cell surface, they were internalized and delivered to lysosomes; chloroquine inhibited subsequent proteolysis of LDL and thereby increased the cellular content of the particles. A physiological oleate to albumin molar ratio, i.e., 1:1 (25 microM oleate and 2 mg/mL albumin), was sufficient to significantly (p less than 0.01) activate all three steps of this alternate pathway: for example, 644 +/- 217 (25 microM oleate) versus 33 +/- 57 (no oleate) ng of LDL/mg of cell protein was degraded after incubation (2 h, 37 degrees C) with 50 micrograms/mL 125I-LDL. We speculate that this pathway could contribute to the clearance of both chylomicron remnants and LDL.
Prothrombinase complex assembly. Kinetic mechanism of enzyme assembly on phospholipid vesicles. (1988)

The kinetics of the assembly of the prothrombinase complex was studied using factor Xa modified with the active site-directed fluorophore dansylglutamylglycylarginyl chloromethyl ester (DEGR.Xa) as a reporter for the process. Stopped-flow kinetic studies of prothrombinase assembly were undertaken at saturating concentrations of calcium ion, using vesicles composed of phosphatidylcholine and phosphatidylserine (PCPS), factor Va, and DEGR.Xa. The rate of complex formation, studied under pseudo first-order conditions, depended on the premixing protocol used to initiate complex assembly and was most rapid when prothrombinase assembly was initiated by reacting Va with the preformed DEGR.Xa.PCPS complex. Under these conditions, reaction rate was linearly dependent on the concentration of PCPS with no evidence for saturation and was independent of the concentration of VA. Essentially identical reaction rates were observed when the association of Va with PCPS was independently assessed by stopped-flow light scattering. The data indicate that prothrombinase assembly initiated by mixing Va with the DEGR.Xa.PCPS complex is rate limited by the initial reaction between Va and available combining sites on the vesicle surface (k = 5.7 x 10(7) M-1.s-1) followed by extremely rapid reactions (k greater than 1 x 10(9) M-1.s-1) between PCPS-bound DEGR.Xa and PCPS-bound Va. The formation of a separate Va.PCPS complex is therefore required for prothrombinase assembly to proceed. Reaction rates obtained when prothrombinase assembly was initiated by reacting DEGR.Xa with the preformed Va.PCPS complex or by reacting PCPS with a mixture of DEGR.Xa and Va were lower. Under these initiation conditions, reaction rates were inhibited by increasing concentrations of factor Va. These data indicate that the formation of the DEGR.Xa.PCPS binary complex is also a prerequisite for prothrombinase assembly. The second-order rate constant for the reaction between factor Xa and PCPS (k = 2.9 x 10(7) M-1.s-1) and the first-order rates of dissociation of the Va.PCPS complex (koff = 0.17 s-1) and the Xa.PCPS complex (koff = 3.3 s-1) were independently determined by stopped-flow light scattering. Collectively, the data are consistent with the interpretation that prothrombinase assembly proceeds via the initial formation of separate Xa.PCPS and Va. PCPS binary complexes at near diffusion-limited rates on the same vesicle. The lipid-bound constituents then react rapidly via rearrangements on the vesicle surface to form prothrombinase.
Reversible anergy of sIgM-mediated signaling in the two subsets of CLL defined by VH-gene mutational status (2007)

The 2 subsets of chronic lymphocytic leukemia (CLL), of worse or better prognosis, likely derive from pre-GC unmutated B cells, or post-GC mutated B cells, respectively. Different clinical behavior could relate to the ability of tumor cells to respond to surface (sIg)-mediated signals. Unmutated cases (U-CLL) have an increased ability to phosphorylate p72(Syk) in response to sIgM ligation compared to mutated cases (M-CLL). We now confirm and further investigate this differential signaling in a large cohort by [Ca(2+)](i) mobilization. Cases responding to sIgM ligation express higher levels of CD38, ZAP-70, and sIgM. However, CD38 does not influence signaling in vitro or associate with response in bimodal CD38-expressing cases. Similarly, ZAP-70 expression is not required for response in either U-CLL or M-CLL. Strikingly, partially or completely anergized sIgM responses from each subset can recover both sIgM expression and signal capacity spontaneously in vitro or following capping/endocytosis. This provides direct evidence for engagement of putative antigen in vivo. Signaling via sIgD differs markedly being almost universally positive in both U-CLL and M-CLL, with no association with CD38 or ZAP-70 expression. Downstream signaling pathways, therefore, appear intact in CLL, locating anergy to sIgM, mainly in M-CLL. Integration of differential isotype-specific effects mediated by (auto)antigen may determine tumor behavior.
Protein Adsorption Kinetics Drastically Altered by Repositioning a Single Charge (1995)

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTProtein Adsorption Kinetics Drastically Altered by Repositioning a Single ChargeJ. J. Ramsden, D. J. Roush, D. S. Gill, R. Kurrat, and R. C. WillsonCite this: J. Am. Chem. Soc. 1995, 117, 33, 8511–8516Publication Date (Print):August 1, 1995Publication History Published online1 May 2002Published inissue 1 August 1995https://pubs.acs.org/doi/10.1021/ja00138a003https://doi.org/10.1021/ja00138a003research-articleACS PublicationsRequest reuse permissionsArticle Views232Altmetric-Citations68LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq (2016)

We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on- and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many false-positive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome.
Spatial memory is related to hippocampal subcellular concentrations of calcium-dependent protein kinase C isoforms in young and aged rats (1997)

Relationships were examined between spatial learning and hippocampal concentrations of the alpha, beta2, and gamma isoforms of protein kinase C (PKC), an enzyme implicated in neuronal plasticity and memory formation. Concentrations of PKC were determined for individual 6-month-old (n = 13) and 24-month-old (n = 27) male Long-Evans rats trained in the water maze on a standard place-learning task and a transfer task designed for rapid acquisition. The results showed significant relationships between spatial learning and the amount of PKC among individual subjects, and those relationships differed according to age, isoform, and subcellular fraction. Among 6-month-old rats, those with the best spatial memory were those with the highest concentrations of PKCgamma in the particulate fraction and of PKCbeta2 in the soluble fraction. Aged rats had increased hippocampal PKCgamma concentrations in both subcellular fractions in comparison with young rats, and memory impairment was correlated with higher PKCgamma concentrations in the soluble fraction. No age difference or correlations with behavior were found for concentrations of PKCgamma in a comparison structure, the neostriatum, or for PKCalpha in the hippocampus. Relationships between spatial learning and hippocampal concentrations of calcium-dependent PKC are isoform-specific. Moreover, age-related spatial memory impairment is associated with altered subcellular concentrations of PKCgamma and may be indicative of deficient signal transduction and neuronal plasticity in the hippocampal formation.
Directed Evolution and Biocatalysis (2001)

This review describes the current state of biocatalysis in the chemical industry. Although we recognize the advantages of chemical approaches, we suggest that the use of biological catalysis is about to expand dramatically because of the recent developments in the artificial evolution of genes that code for enzymes. For the first time it is possible to consider the rapid development of an enzyme that is designed for a specific chemical reaction. This technology offers the opportunity to adapt the enzyme to the needs of the process. We describe herein the development of enzyme evolution technology and particularly DNA shuffling. We also consider several classes of enzymes, their current applications, and the limitations that should be addressed. In a review of this length it is impossible to describe all the enzymes with potential for industrial exploitation; there are other classes, which given appropriate activity, selectivity, and robustness, could become useful tools for the industrial chemist. This is an exciting era for biocatalysis and we expect great progress in the future.
New Therapeutic Strategies and Drug Candidates for Neurodegenerative Diseases: p53 and TNF‐α Inhibitors, and GLP‐1 Receptor Agonists (2004)

Owing to improving preventative, diagnostic, and therapeutic measures for cardiovascular disease and a variety of cancers, the average ages of North Americans and Europeans continue to rise. Regrettably, accompanying this increase in life span, there has been an increase in the number of individuals afflicted with age-related neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, and stroke. Although different cell types and brain areas are vulnerable among these, each disorder likely develops from activation of a common final cascade of biochemical and cellular events that eventually lead to neuronal dysfunction and death. In this regard, different triggers, including oxidative damage to DNA, the overactivation of glutamate receptors, and disruption of cellular calcium homeostasis, albeit initiated by different genetic and/or environmental factors, can instigate a cascade of intracellular events that induce apoptosis. To forestall the neurodegenerative process, we have chosen specific targets to inhibit that are at pivotal rate-limiting steps within the pathological cascade. Such targets include TNF-alpha, p53, and GLP-1 receptor. The cytokine TNF-alpha is elevated in Alzheimer's disease, Parkinson's disease, stroke, and amyotrophic lateral sclerosis. Its synthesis can be reduced via posttranscriptional mechanisms with novel analogues of the classic drug, thalidomide. The intracellular protein and transcription factor, p53, is activated by the Alzheimer's disease toxic peptide, Abeta, as well as by excess glutamate and hypoxia to trigger neural cell death. It is inactivated by novel tetrahydrobenzothiazole and -oxazole analogues to rescue cells from lethal insults. Stimulation of the glucagon-like peptide-1 receptor (GLP-1R) in brain is associated with neurotrophic functions that, additionally, can protect cells against excess glutamate and other toxic insults.
The Antiinflammatory and Analgesic Effects of Baicalin in Carrageenan-Evoked Thermal Hyperalgesia (2003)

Our results showed that baicalin possesses an analgesic effect in carrageenan-evoked thermal hyperalgesia. The possible mechanisms of action of baicalin may be associated with the inhibition of proinflammatory mediator overproduction, including cytokines, nitric oxide, and prostaglandin E(2), as well as neutrophil infiltration. This implies that baicalin may be a potential therapeutic analgesic for inflammatory pain.
Experimental demonstration of the coexistence of spin Hall and Rashba effects inβ−tantalum/ferromagnetbilayers (2015)

We have measured the spin torques of $\ensuremath{\beta}\text{\ensuremath{-}}\mathrm{tantalum}/\mathrm{C}{\mathrm{o}}_{20}\mathrm{F}{\mathrm{e}}_{60}{\mathrm{B}}_{20}$ bilayers versus Ta thickness at room temperature using a ferromagnetic resonance (FMR) technique. A significant fieldlike spin torque originating from Ta was identified, which is constant and independent of Ta thickness. Because of this constant torque, the spin Hall coefficient ${\ensuremath{\theta}}_{\mathrm{SH}}$ needs to be calculated from the ratio of the symmetric component of the FMR signal to the slope of the antisymmetric component with Ta thickness, from which a value of $\ensuremath{-}0.11\ifmmode\pm\else\textpm\fi{}0.01$ was determined. The saturation magnetization of the CoFeB layers for samples deposited with Ta was found to be smaller than that of a single CoFeB layer, with values of $1.84\ifmmode\pm\else\textpm\fi{}0.01$ and $1.92\ifmmode\pm\else\textpm\fi{}0.01$ T, respectively. The origin of the fieldlike torque is ascribed to an interface spin-orbit coupling, or Rashba effect, due to the strength and constancy of the torque with Ta thickness. From fitting measured data to a semiclassical diffusion model that includes interface spin-orbit coupling, we have determined the spin diffusion length for \ensuremath{\beta}-tantalum to be 2.5 nm.