gebhart/patrepeval huggingface dataset:
Intracellular expression of BPTI fusion proteins and single column cleavage/affinity purification by chymotrypsin (1991)

The novel and efficient expression system described here produces formerly poorly expressed, proteolytically unstable mutants of bovine pancreatic trypsin inhibitor (BPTI). A new, single column method for the cleavage of a recombinant fusion to BPTI and affinity purification of the BPTI moiety by immobilized chymotrypsin is an integral part of the system. Wild-type and mutant BPTI molecules are expressed in Escherichia coli as fusion proteins forming intracellular inclusion bodies. Transcription initiation is under the control of the E. coli trp promoter. The expressed protein is tripartite fusion comprising (i) a portion of the TrpLE leader peptide, (ii) a synthetic IgG binding domain derived from protein A and (iii) the BPTI variant. Solubilization of the inclusion bodies and refolding of the fusion proteins in a thiol-disulfide shuffling system yields correctly folded inhibitor molecules. In the single column purification and cleavage procedure, immobilized chymotrypsin cleaves the refolded fusion protein and releases affinity purified active BPTI mutants with correct N-termini. Mutant BPTI molecules which do not fold into active inhibitors are also stably expressed in inclusion bodies but cannot be purified by this method. Unlike previously described secretion systems for the production of BPTI, expression levels in this system appear to be independent of both the mutation in the BPTI gene and the activity of the expressed protein. Mutants poorly expressed in secretion systems can now be produced in sufficient quantities for protein folding studies and structural analysis using X-ray crystallography and NMR spectroscopy.
Characterization of near-infrared n-type β-FeSi2/p-type Si heterojunction photodiodes at room temperature (2009)

n -type β-FeSi2/p-type Si heterojunctions were fabricated from β-FeSi2 films epitaxially grown on Si(111) by facing-target direct-current sputtering. Sharp film-substrate interfaces were confirmed by scanning electron microscopy. The current-voltage and photoresponse characteristics were measured at room temperature. They exhibited good rectifying properties and a change of approximately one order of magnitude in the current at a bias voltage of −1 V under illumination by a 6 mW, 1.31 μm laser. The estimated detectivity was 1.5×109 cm √Hz W at 1.31 μm. The results suggest that the β-FeSi2/Si heterojunctions can be used as near-infrared photodetectors that are compatible with silicon integrated circuits.
Synthesis and structural investigations of N-alkylated β-peptidosulfonamide–peptide hybrids of the amyloidogenic amylin(20–29) sequence: implications of supramolecular folding for the design of peptide-based bionanomaterials (2006)

The incorporation of a single beta-aminoethane sulfonyl amide moiety in a highly amyloidogenic peptide sequence resulted in a complete loss of amyloid fibril formation. Instead, supramolecular folding morphologies were observed. Subsequent chemoselective N-alkylation of the sulfonamide resulted in amphiphilic peptide-based hydrogelators. It was found that variation of merely the alkyl chain induced a dramatic variation in aggregation motifs such as helical ribbons and tapes, ribbons progressing to closed tubes, twisted lamellar sheets and entangled/branched fibers.
Large‐scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP‐fusion genomic DNA library (2000)

A number of new intracellular structural components were found in this library. The library of GFP-fusion constructs also provides useful fluorescent markers for various intracellular structures and cellular activities, which can be readily used for microscopic observation in living cells.
Bacteriophage lambda surface display of a bacterial biotin acceptor domain reveals the minimal peptide size required for biotinylation (1998)

Phage display is a powerful technique for identifying specific ligands to a given target. In this work random peptides derived from the biotin accepting domain of the Klebsiella pneumoniae oxaloacetate decarboxylase were displayed on bacteriophage lambda heads to determine the minimal sequence length that is necessary to effect biotinylation in vivo. Phages with a functional biotinylation domain were identified after affinity purification with immobilised avidin. All biotinylated phages isolated this way were found to have a sequence of 66 amino acids from the parental protein in common. This minimal biotinylation domain is fully functional as a biotin acceptor and more resistant to proteolytic attack compared to domains of larger size derived from the same protein. The data present the first example of a posttranslational protein modification analysed in a phage display system. Moreover, a biotin domain of reduced size and improved stability was identified, that should be superior to the larger parental protein as a tag to generate biotinylated fusion proteins.
The Functional Diffusion Map: An Imaging Biomarker for the Early Prediction of Cancer Treatment Outcome (2006)

Functional diffusion map (fDM) has been recently reported as an early and quantitative biomarker of clinical brain tumor treatment outcome. This approach spatially maps and quantifies treatment-induced changes in tumor water diffusion values resulting from alterations in cell density/cell membrane function and microenvironment. This current study was designed to evaluate the capability of fDM for preclinical evaluation of dose escalation studies and to determine if these changes were correlated with outcome measures (cell kill and overall survival). Serial T2-weighted were carried out on rodents with orthotopically implanted 9L brain tumors receiving three doses of 1,3-bis(2-chloroethyl)-1-nitrosourea (6.65, 13.3, and 26.6 mg/kg, i.p.). All images were coregistered to baseline T2-weighted images for fDM analysis. Analysis of tumor fDM data on day 4 posttreatment detected dose-dependent changes in tumor diffusion values, which were also found to be spatially dependent. Histologic analysis of treated tumors confirmed spatial changes in cellularity as observed by fDM. Early changes in tumor diffusion values were found to be highly correlative with drug dose and independent biologic outcome measures (cell kill and survival). Therefore, The fDM imaging biomarker for early prediction of treatment efficacy can be used in the drug development process.
Calibration requirements and procedures for a monitor-based augmented reality system (1995)

Augmented reality entails the use of models and their associated renderings to supplement information in a real scene. In order for this information to be relevant or meaningful, the models must be positioned and displayed in such a way that they blend into the real world in terms of alignments, perspectives, illuminations, etc. For practical reasons the information necessary to obtain this realistic blending cannot be known a priori, and cannot be hard wired into a system. Instead a number of calibration procedures are necessary so that the location and parameters of each of the system components are known. We identify the calibration steps necessary to build a computer model of the real world and then, using the monitor based augmented reality system developed at ECRC (GRASP) as an example, we describe each of the calibration processes. These processes determine the internal parameters of our imaging devices (scan converter, frame grabber, and video camera), as well as the geometric transformations that relate all of the physical objects of the system to a known world coordinate system.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>
A Convenient Method for Synthesis of Enantiomerically Enriched Methylphenidate from N-Methoxycarbonylpiperidine (1999)

[formula: see text] This report describes a new method to prepare optically active methylphenidate starting from piperidine. The method consists of a transformation of N-methoxycarbonylpiperidine to the corresponding alpha-methoxylated carbamate I by utilizing electrochemical oxidation followed by the coupling reaction with optically active Evans imides II to produce optically active methylphenidate derivatives III with high stereoselectivities, threo-(2R,2'R)-Methylphenidate (IV; Ar = Ph; Ritalin) was easily prepared from III in three steps.
Applications of two-photon fluorescence microscopy in deep-tissue imaging (2000)

Based on the non-linear excitation of fluorescence molecules, two-photon fluorescence microscopy has become a significant new tool for biological imaging. The point-like excitation characteristic of this technique enhances image quality by the virtual elimination of off-focal fluorescence. Furthermore, sample photodamage is greatly reduced because fluorescence excitation is limited to the focal region. For deep tissue imaging, two-photon microscopy has the additional benefit in the greatly improved imaging depth penetration. Since the near- infrared laser sources used in two-photon microscopy scatter less than their UV/glue-green counterparts, in-depth imaging of highly scattering specimen can be greatly improved. In this work, we will present data characterizing both the imaging characteristics (point-spread-functions) and tissue samples (skin) images using this novel technology. In particular, we will demonstrate how blind deconvolution can be used further improve two-photon image quality and how this technique can be used to study mechanisms of chemically-enhanced, transdermal drug delivery.
CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells (2021)

CRISPR-Cas9 cytidine and adenosine base editors (CBEs and ABEs) can disrupt genes without introducing double-stranded breaks by inactivating splice sites (BE-splice) or by introducing premature stop (pmSTOP) codons. However, no in-depth comparison of these methods or a modular tool for designing BE-splice sgRNAs exists. To address these needs, we develop SpliceR ( http://z.umn.edu/spliceR ) to design and rank BE-splice sgRNAs for any Ensembl annotated genome, and compared disruption approaches in T cells using a screen against the TCR-CD3 MHC Class I immune synapse. Among the targeted genes, we find that targeting splice-donors is the most reliable disruption method, followed by targeting splice-acceptors, and introducing pmSTOPs. Further, the CBE BE4 is more effective for disruption than the ABE ABE7.10, however this disparity is eliminated by employing ABE8e. Collectively, we demonstrate a robust method for gene disruption, accompanied by a modular design tool that is of use to basic and translational researchers alike.
Promising preclinical activity of 2-methoxyestradiol in multiple myeloma (2003)

2ME2 has significant activity against myeloma cell lines and primary myeloma cells both in vitro and in an animal model. Clinical trials are required to evaluate its activity in patients with MM.
The BRCT Domain Is a Phospho-Protein Binding Domain (2003)

The carboxyl-terminal domain (BRCT) of the Breast Cancer Gene 1 (BRCA1) protein is an evolutionarily conserved module that exists in a large number of proteins from prokaryotes to eukaryotes. Although most BRCT domain-containing proteins participate in DNA-damage checkpoint or DNA-repair pathways, or both, the function of the BRCT domain is not fully understood. We show that the BRCA1 BRCT domain directly interacts with phosphorylated BRCA1-Associated Carboxyl-terminal Helicase (BACH1). This specific interaction between BRCA1 and phosphorylated BACH1 is cell cycle regulated and is required for DNA damage-induced checkpoint control during the transition from G2 to M phase of the cell cycle. Further, we show that two other BRCT domains interact with their respective physiological partners in a phosphorylation-dependent manner. Thirteen additional BRCT domains also preferentially bind phospho-peptides rather than nonphosphorylated control peptides. These data imply that the BRCT domain is a phospho-protein binding domain involved in cell cycle control.
Studies on the Metabolism of D- and L-Isomers of 3, 4-Dihydroxyphenylalanine (DOPA). V. Mechanism of Intestinal Absorption of D- and L-DOPA-<SUP>14</SUP>C in Rats (1973)

The rate and mechanism of intestinal absorption of D- and L-isomers of 3, 4-dihydroxyphenylalanine (DOPA) were comparatively investigated in rats by means of in situ ligated loop and in vitro tissue accumulation techniques. L-DOPA was found to be very easily and much more rapidly absorbed from the ligated loop of rat intestine than the D-isomer and the absorption was proved to follow saturation kinetics and to be inhibited competitively by L-alanine and L-phenylalanine, while not by D-phenylalanine and D-DOPA. The in vitro tissue uptake of L-DOPA-14C revealed an accumulation of radioactivity against the concentration gradient, which was depressed by metabolic inhibitors as DNP and sodium azide. L-DOPA was extensively metabolized in the intestinal tissue to dopamine, its glucuronide and DOPAC, while the D-isomer was not metabolized to any appreciable extent. The rate of absorption and the tissue uptake of L-DOPA-14C was, however, not affected by inhibition of DOPA-decarboxylase. A saturability of the intestinal DOPA-decarboxylase activity with the substrate was demonstrated by everted sac method with increasing L-DOPA concentration in the incubation medium. From these results, it was concluded that L-DOPA, but not D-DOPA, is absorbed from rat intestine by an active transport mechanism, which proceeds independently from the decarboxylation process. These differences between the two isomers were also discussed in relation to those in their distribution patterns after oral administration to rats.
Exploring light propagating in photonic crystals with Fourier optics (2007)

Photonic crystals (PhCs) act on light in two different ways: confinement and modification of propagation. Both phenomena rely on the complex interplay between multiply scattered waves that can form what is known as a Bloch mode. Here, we present a technique that allows direct imaging of Bloch modes, both in real space and in k-space. The technique gives access to the location of the field maxima inside the PhC, the dispersion relation, equifrequency surfaces, as well as reflection and transmission coefficients. Our key advance is that we retrieve the desired information comprehensively, without postprocessing or cumbersome near-field scanning techniques, even for modes that are nominally lossless, i.e., below the light line. To highlight the potential of the technique, we extract the dispersion curve of a coupled cavity waveguide consisting of as many as 100 cavities, as well as the equifrequency surfaces and polarization properties of a PhC beam splitter.
Photocleavable DNA Barcode–Antibody Conjugates Allow Sensitive and Multiplexed Protein Analysis in Single Cells (2012)

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.
An ATP-binding cassette transporter-like complex governs cell-wall hydrolysis at the bacterial cytokinetic ring (2011)

ATP-binding cassette transporters are ubiquitous membrane protein complexes that move substrates across membranes. They do so using ATP-induced conformational changes in their nucleotide-binding domains to alter the conformation of the transport cavity formed by their transmembrane domains. In Escherichia coli, an ATP-binding cassette transporter-like complex composed of FtsE (nucleotide-binding domain) and FtsX (transmembrane domain) has long been known to be important for cytokinesis, but its role in the process has remained mysterious. Here we identify FtsEX as a regulator of cell-wall hydrolysis at the division site. Cell-wall material synthesized by the division machinery is shared initially by daughter cells and must be split by hydrolytic enzymes called "amidases" to drive daughter-cell separation. We recently showed that the amidases require activation at the cytokinetic ring by proteins with LytM domains, of which EnvC is the most critical. In this report, we demonstrate that FtsEX directly recruits EnvC to the septum via an interaction between EnvC and a periplasmic loop of FtsX. Importantly, we also show that FtsEX variants predicted to be ATPase defective still recruit EnvC to the septum but fail to promote cell separation. Our results thus suggest that amidase activation via EnvC in the periplasm is regulated by conformational changes in the FtsEX complex mediated by ATP hydrolysis in the cytoplasm. Since FtsE has been reported to interact with the tubulin-like FtsZ protein, our model provides a potential mechanism for coupling amidase activity with the contraction of the FtsZ cytoskeletal ring.
Exploring the Validity of Valproic Acid Animal Model of Autism (2015)

The valproic acid (VPA) animal model of autism spectrum disorder (ASD) is one of the most widely used animal model in the field. Like any other disease models, it can't model the totality of the features seen in autism. Then, is it valid to model autism? This model demonstrates many of the structural and behavioral features that can be observed in individuals with autism. These similarities enable the model to define relevant pathways of developmental dysregulation resulting from environmental manipulation. The uncovering of these complex pathways resulted to the growing pool of potential therapeutic candidates addressing the core symptoms of ASD. Here, we summarize the validity points of VPA that may or may not qualify it as a valid animal model of ASD.
Quantitative modeling of inland water quality for high-resolution MSS systems (1991)

For a lake system in The Netherlands, Secchi disk transparency, seston dry weight, vertical attenuation coefficients, chlorophyll-a, and phaeopigments were determined. Inherent optical properties were estimated from measured apparent optical properties. Subsurface spectroradiometric measurements and airborne (non)-imaging spectrometric measurements were performed. This made it possible to model the performance of high-resolution multispectral scanning systems. Results of multivariate statistical modeling of water quality parameters versus simulated spectral band ratios for a water quality spectral bandset of the programmable multispectral imager (PMI), also known as the fluorescence line imager (FLI), are presented.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>
Self-administered topical imiquimod treatment of vulvar intraepithelial neoplasia. A report of four cases. (2000)

Imiquimod may be an effective treatment modality for viral VIN 3 in the future.
Homology Directed Knockin of Point Mutations in the Zebrafish tardbp and fus Genes in ALS Using the CRISPR/Cas9 System (2016)

The methodology for site-directed editing of single nucleotides in the vertebrate genome is of considerable interest for research in biology and medicine. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 type II (Cas9) system has emerged as a simple and inexpensive tool for editing genomic loci of interest in a variety of animal models. In zebrafish, error-prone non-homologous end joining (NHEJ) has been used as a simple method to disrupt gene function. We sought to develop a method to easily create site-specific SNPs in the zebrafish genome. Here, we report simple methodologies for using CRISPR/Cas9-mediated homology directed repair using single-stranded oligodeoxynucleotide donor templates (ssODN) for site-directed single nucleotide editing, for the first time in two disease-related genes, tardbp and fus.
Novel Iso-branched Ether Lipids as Specific Markers of Developmental Sporulation in the Myxobacterium Myxococcus xanthus (2006)

Iso-fatty acids (FAs) are the dominant FA family in all myxobacteria analyzed. Furthermore, it was postulated that iso-FAs or compounds derived thereof are involved in fruiting body formation in Myxococcus xanthus, since mutants with a reduced level of iso-FA due to a reduced level of the precursor isovaleryl-CoA, are delayed in aggregation and produce only few myxospores. To elucidate the function of iso-FAs and their corresponding lipids we have analyzed the developmental phenotype of mutants having different levels of iso-FAs resulting in a clear correlation between the amount of iso-FAs and the delay of aggregation and reduction in spore yield. Addition of either isovalerate or 13-methyltetradecanoic acid resulted in restoration of the wild-type FA profile and normal development. Detailed analysis of the fatty acid (FA) profile during fruiting body formation in Myxococcus xanthus wild-type revealed the specific accumulation of 13-methyltetradecanal and 1-O-13-methyltetradecylglycerol which were produced specifically in the myxospores and which are derived from 1-O-(13-methyl-1-Z-tetradecenyl)-2-O-(13-methyltetradecanoyl)-glycero-3-phosphatidylethanolamine (VEPE) and 1,2-di-(13-methyltetradecanoyl)-3-(13-methyltetradecyl)glycerol (TG-1), respectively. The structures of these unusual ether lipids have been determined by spectrometric methods and synthesis (for TG-1). Analysis of several mutants blocked at different stages of development indicated that the biosynthesis of TG-1 is developmentally regulated and that VEPE might be an intermediate in the TG-1 biosynthesis. Finally, addition of TG-1 to mutants blocked in the biosynthesis of isovaleryl-CoA could restore aggregation and sporulation emphasizing the important role of iso-branched lipids for myxobacterial development.
The hexokinase 2 protein regulates the expression of the GLK1, HXK1 and HXK2 genes of Saccharomyces cerevisiae (2001)

The key glycolytic HXK2 gene, coding for the enzyme hexokinase 2 (Hxk2p), is expressed when cells of the yeast Saccharomyces cerevisiae are grown on a fermentable medium using glucose, fructose or mannose as a carbon source. After shifting the cells to a non-fermentable carbon source, the HXK2 gene is repressed and the HXK1 and GLK1 genes are rapidly de-repressed, producing the enzymes hexokinase 1 (Hxk1p) and glucokinase (Glk1p) respectively. Because the in vivo functions of the Hxk1p and Glk1p enzymes have remained a mystery so far, we have investigated this glucose-induced regulatory process. Here we demonstrate the involvement of Hxk2p in the glucose-induced repression of the HXK1 and GLK1 genes and the glucose-induced expression of the HXK2 gene. We have also demonstrated the involvement of Hxk1p as a negative factor in the expression of the GLK1 and HXK2 genes. Further experimental evidence, using mutant cells expressing a truncated version of Hxk2p unable to enter the nucleus, shows that nuclear localization of Hxk2p is necessary for glucose-induced repression signalling of the HXK1 and GLK1 genes and for glucose-induced expression of the HXK2 gene. Gel mobility-shift analysis shows that Hxk2p-mediated regulation is exerted through ERA (ethanol repression autoregulation)-like regulatory sequences present in the HXK1 and GLK1 promoters and in two downstream repressing sequences of the HXK2 gene. These findings reveal a novel mechanism of gene regulation whereby the product of a glycolytic gene, normally resident in the cytosol, interacts directly with nuclear proteins to regulate the transcription of the HXK1 and GLK1 genes and to autoregulate its own transcription.