Interrogating Precision Electrophile Signaling.

Understanding the targets and signaling roles of reactive electrophilic species (RES) at a specific cellular space and time has long been hampered by the reliance of the field on the bulk administration of excess RES from outside of cells and/or animals. Uncontrolled bolus methods provide limited understanding of target engagement for these individual nonenzymatic RES-modification events. REX technologies [targetable reactive electrophiles and oxidants (T-REX) and its genome-wide variant (G-REX)] were developed as a gateway to address these limitations. These protocols offer a new ability to both profile kinetically privileged sensors (KPSs) of RES at a systems level (G-REX™ profiling) and monitor signaling responses at the sensor protein-of-interest (POI)-specific level (T-REX™ delivery) with high spatiotemporal resolution. REX technologies are compatible with several model systems and are built on a HaloTag-targetable small-molecule photocaged precursor to a native RES.

Inducible Genetic Code Expansion in Eukaryotes.

Genetic code expansion (GCE) is a versatile tool to site-specifically incorporate a noncanonical amino acid (ncAA) into a protein, for example, to perform fluorescent labeling inside living cells. To this end, an orthogonal aminoacyl-tRNA-synthetase/tRNA (RS/tRNA) pair is used to insert the ncAA in response to an amber stop codon in the protein of interest. One of the drawbacks of this system is that, in order to achieve maximum efficiency, high levels of the orthogonal tRNA are required, and this could interfere with host cell functionality. To minimize the adverse effects on the host, we have developed an inducible GCE system that enables us to switch on tRNA or RS expression when needed. In particular, we tested different promotors in the context of the T-REx or Tet-On systems to control expression of the desired orthogonal tRNA and/or RS. We discuss our result with respect to the control of GCE components as well as efficiency. We found that only the T-REx system enables simultaneous control of tRNA and RS expression.

Effect of mTOR inhibitors on sodium taurocholate cotransporting polypeptide (NTCP) function in vitro.

The sodium taurocholate cotransporting polypeptide (NTCP; gene name SLC10A1) is the primary hepatic basolateral uptake transporter for conjugated bile acids and the entry receptor for the hepatitis B and D virus (HBV/HDV). Regulation of human NTCP remains a knowledge gap due to significant species differences in substrate and inhibitor selectivity and plasma membrane expression. In the present study, various kinase inhibitors were screened for inhibition of NTCP function and taurocholate (TCA) uptake using NTCP-transfected HuH-7 cells. This study identified everolimus, an mTOR inhibitor and macrocyclic immunosuppressive drug, as an NTCP inhibitor with modest potency (IC50 = 6.7-8.0 µM). Further investigation in differentiated HuH-7 cells expressing NTCP and NTCP-overexpressing Flp-In T-REx 293 cells revealed that the mechanism of action of everolimus on NTCP is direct inhibition and mTOR-independent. Structural analogs of everolimus inhibited NTCP-mediated TCA uptake, however, functional analogs did not affect NTCP-mediated TCA transport, providing further evidence for direct inhibition. This work contributes to the growing body of literature suggesting that NTCP-mediated bile acid uptake may be inhibited by macrocyclic peptides, which may be further exploited to develop novel medications against HBV/HDV.

Employing the Operational Model to Measure System-Independent OTR Efficacy.

Ideally, pharmacological analysis is based on quantitative measurements applied toward unveiling biological mechanisms and guiding medicinal chemistry efforts in drug discovery and basic research. The most robust analysis frameworks generate findings which can be dissociated from the specific experimental setting to provide insights of a broader scope. With insufficient efficacy being the leading cause of drug failures in late-stage clinical trials, more rigorous preclinical definitions might assist in better translation. This chapter details a new method for accessing the Black and Leff operational model of agonism using a stable Flp-In™ T-REx™ HEK293 cell line under tetracycline-dependent control of OTR expression. We cover steps for performing the Gq-coupled HTRF® IP-One assay, curve-fitting data, calculating and statistically representing system-independent drug activity, predicting responses in different sensitivities, and plotting of multivariate analyses.

Investigation of sonochemical treatment of heavy hydrocarbon by ultrasound-assisted cavitation.

A highly viscous nature of heavy oil poses challenges to transportation leading to costly operation and difficult processing. Traditional methods of upgrading unconventional hydrocarbon sources involve catalytic and thermal upgrading and these methods require high temperature and pressure. In the present study, partial upgrading of heavy hydrocarbon is studied by using cavitation and the stimulator. Cavitation is a phenomenon comprising of formation, growth and collapse of bubbles in a liquid medium. The most well-known disruptive effect of cavitation occurs during the collapse phase of bubbles. Method of inducing cavitation involves transmitting 20 kHz of ultrasound through an ultrasonic horn. A model molecule used in this study is n-hexadecane (C16). The experiments were carried out at 230 °C, atmospheric pressure and 60 min time scale. The results indicated that the conversion of n-hexadecane into R1 fraction (C16) was 4.46% for the cavitation-assisted cracking with the stimulator. The selectivity to R1 and R2 fractions were 71% and 29%, respectively. Adding 5 vol% decalin as hydrogen donor into the cracking process yielded 9.18% conversion of n-hexadecane into R1 and R2 fractions. In addition, the selectivity to R1 and R2 fractions were 87% and 13%. This study focuses on less energy intensive process for heavy hydrocarbon by utilizing cavitation and the stimulator and how ultrasound-assisted cracking with the stimulator could be a viable alternative to treat heavy hydrocarbon at the low temperature.

REX technologies for profiling and decoding the electrophile signaling axes mediated by Rosetta Stone proteins.

It is now clear that some cysteines on some proteins are highly tuned to react with electrophiles. Based on numerous studies, it is also established that electrophile sensing underpins rewiring of several critical signaling processes. These electrophile-sensing proteins, or privileged first responders (PFRs), are likely critically relevant for drug design. However, identifying PFRs remains a challenging and unsolved problem, despite the development of several high-throughput methods to ID proteins that react with electrophiles. More importantly, we remain unable to rank how different PFRs identified under different conditions relate to one another, in terms of sensing or signaling capacity. Here we evaluate different methods to assay sensing functions of proteins and discuss these methods in the context of developing a "ranking scheme." Based on theoretical and experimental evidence, we propose that T-REX-the only targeted-electrophile delivery tool presently available-is a reliable method to rank PFRs. Finally, we address to what extent electrophile sensing and downstream signaling are correlated. Based on our current data, we observe that such behaviors are indeed correlated. It is our hope that through this manuscript researchers from various arms of the stress signaling fields will focus on developing a quantitative understanding of precision electrophile labeling.

Richter's syndrome of T-cell lineage (T rex lymphoma).

By definition, Richter's syndrome represents the transformation of low-grade B-cell lymphoma into high-grade B-cell lymphoma, usually refractory to treatment. Exceptional cases of transformation into very aggressive mature T-cell lymphomas have been described as an unusual manifestation of the syndrome in patients died after few months from the diagnosis, despite chemotherapy. The time is ripe to regroup these T lymphomas under a new pathological subset, through the unequivocal alternate naming of 'T rex lymphoma', by analogy with the aggressive behavior of the famous dinosaur (T. rex). In practice, it represents the transformation of low-grade B-cell lymphoma into high-grade T-cell lymphoma, burdened by a very poor prognosis, because of the underlying B-cell lymphoma, which negatively interferes with the immune response of the patient. Against this distinct lymphomatous T clone, the major therapeutic efforts should be addressed.

Chemical Biology Gateways to Mapping Location, Association, and Pathway Responsivity.

Here we discuss, how by applying chemical concepts to biological problems, methods have been developed to map spatiotemporal regulation of proteins and small-molecule modulation of proteome signaling responses. We outline why chemical-biology platforms are ideal for such purposes. We further discuss strengths and weaknesses of chemical-biology protocols, contrasting them against classical genetic and biochemical approaches. We make these evaluations based on three parameters: occupancy; functional information; and spatial restriction. We demonstrate how the specific choice of chemical reagent and experimental set-up unite to resolve biological problems. Potential improvements/extensions as well as specific controls that in our opinion are often overlooked or employed incorrectly are also considered. Finally, we discuss some of the latest emerging methods to illuminate how chemical-biology innovations provide a gateway toward information hitherto inaccessible by conventional genetic/biochemical means. Finally, we also caution against solely relying on chemical-biology strategies and urge the field to undertake orthogonal validations to ensure robustness of results.

A closed formula for calculating pesticide residue levels in the feed items of terrestrial species.

Calculating pesticide residue levels in feed items for terrestrial species requires accounting for the application rate of the pesticide, the frequency and interval of application, the half-life of the pesticide on the food item, and the residue unit dose. Microsoft Excel™-based applications such as the US Environmental Protection Agency's Terrestrial Residue Exposure model (T-REX) and Terrestrial Herpetofaunal Exposure Residue Program (T-HERPS) calculate the residue levels in feed items using a recursive sequence. A recursive sequence is an unwieldy calculation method that presents a barrier to creating a software-based tool capable of conducting flexible assessments. Therefore, we determined the closed form of the recursive mathematical equation used by both T-REX and T-HERPS. With this formula, we can both duplicate screening-level assessments (T-REX, T-HERPS) as well as incrementally refine the assessment with data-driven inputs. Integr Environ Assess Manag 2018;14:703-709. © 2018 SETAC.

Single-Protein-Specific Redox Targeting in Live Mammalian Cells and C. elegans.

T-REX (targetable reactive electrophiles and oxidants) enables electrophile targeting in living systems with high spatiotemporal precision and at single-protein-target resolution. T-REX allows functional consequences of individual electrophile signaling events to be directly linked to on-target modifications. T-REX is accomplished by expressing a HaloTagged protein of interest (POI) and introducing a Halo-targetable bioinert photocaged precursor to a reactive electrophilic signal (RES). Light exposure releases the unfettered RES on demand, enabling precision modification of the POI due to proximity. Using alkyne-functionalized 4-hydroxynonenal (HNE) as a representative RES, this protocol delineates optimized strategies to (1) execute T-REX in live human cells and C. elegans, (2) quantitate the POI's RES-sensitivity by either azido-fluorescent-dye conjugation or (3) enrich using biotin-azide/streptavidin pulldown procedure in both model systems, and (4) identify the site of RES-labeling on the POI using proteomics. Built-in T-REX controls that allow users to directly confirm on-target/on-site specificity of RES-sensing are also described. © 2018 by John Wiley & Sons, Inc.

MimiLook: A Phylogenetic Workflow for Detection of Gene Acquisition in Major Orthologous Groups of Megavirales.

With the inclusion of new members, understanding about evolutionary mechanisms and processes by which members of the proposed order, Megavirales, have evolved has become a key area of interest. The central role of gene acquisition has been shown in previous studies. However, the major drawback in gene acquisition studies is the focus on few MV families or putative families with large variation in their genetic structure. Thus, here we have tried to develop a methodology by which we can detect horizontal gene transfers (HGTs), taking into consideration orthologous groups of distantly related Megavirale families. Here, we report an automated workflow MimiLook, prepared as a Perl command line program, that deduces orthologous groups (OGs) from ORFomes of Megavirales and constructs phylogenetic trees by performing alignment generation, alignment editing and protein-protein BLAST (BLASTP) searching across the National Center for Biotechnology Information (NCBI) non-redundant (nr) protein sequence database. Finally, this tool detects statistically validated events of gene acquisitions with the help of the T-REX algorithm by comparing individual gene tree with NCBI species tree. In between the steps, the workflow decides about handling paralogs, filtering outputs, identifying Megavirale specific OGs, detection of HGTs, along with retrieval of information about those OGs that are monophyletic with organisms from cellular domains of life. By implementing MimiLook, we noticed that nine percent of Megavirale gene families (i.e., OGs) have been acquired by HGT, 80% OGs were Megaviralespecific and eight percent were found to be sharing common ancestry with members of cellular domains (Eukaryote, Bacteria, Archaea, Phages or other viruses) and three percent were ambivalent. The results are briefly discussed to emphasize methodology. Also, MimiLook is relevant for detecting evolutionary scenarios in other targeted phyla with user defined modifications. It can be accessed at following link 10.6084/m9.figshare.4653622.

Cavitands Endow All-Dielectric Beads With Selectivity for Plasmon-Free Enhanced Raman Detection of Nε-Methylated Lysine.

SiO2/TiO2 microbeads (T-rex) are promising materials for plasmon-free surface-enhanced Raman scattering (SERS), offering several key advantages in biodiagnostics. In this paper we report the combination of T-rex beads with tetraphosphonate cavitands (Tiiii), which imparts selectivity toward Nε-methylated lysine. SERS experiments demonstrated the efficiency and selectivity of the T-rex-Tiiii assays in detecting methylated lysine hydrochloride (Nε-Me-Lys-Fmoc) from aqueous solutions, even in the presence of the parent Lys-Fmoc hydrochloride as interferent. The negative results obtained in control experiments using TSiiii ruled out any other form of surface recognition or preferential physisorption. MALDI-TOF analyses on the beads exposed to Nε-Me-Lys-Fmoc revealed the presence of the Tiiii•Nε-Me-Lys-Fmoc complex. Raman analyses based on the intensity ratio of Nε-Me-Lys-Fmoc and cavitand-specific modes resulted in a dose-response plot, which allowed for estimating the concentration of Nε-methylated lysine from initial solutions in the 1 × 10(-3) to 1 × 10(-5) M range. These results can set the basis for the development of new Raman assays for epigenetic diagnostics.

Using the terrestrial residue exposure (T-REX) model to assess threatened and endangered bird exposure to and risk from pesticides.

The Terrestrial Residue Exposure (T-REX) model, a spreadsheet-based model developed by the US Environmental Protection Agency (USEPA), is used to estimate the concentrations of pesticides on some representative avian terrestrial food items after a foliar pesticide application. T-REX uses 6 categories of food items to assess exposure for birds. Different body size classes are used to estimate diet intake levels and to demonstrate different exposures for different size birds with various single component diets. The purpose of this analysis is to determine whether T-REX is a sufficient tool to assess exposure to the majority of threatened and endangered (T&E) bird species in the United States based on diets and body sizes of listed species. Our analysis combining diets and body weights of T&E species finds that no listed species weighing less than 50 g consumes primarily plant matter. Therefore, the hypothetical bird included in T-REX weighing 20 g and consuming only short grass that is predicted to have the highest exposure does not represent any T&E bird species. Many T&E species are represented when T-REX considers risk for insectivorous species in the 20-g size class. However, no T&E species that are predominantly insectivorous occur in the larger 100 and 1000-g size classes. Fruit and seed-eating T&E species occur in each of the size classes that T-REX considers, so fruit and seed-eating T&E birds are adequately represented in T-REX. T-REX does not include any estimated environmental concentrations (EECs) for aquatic dietary items or nectar, terrestrial vertebrates, soil-dwelling organisms, and noninsect invertebrates (e.g., snails). More than a third of T&E species have diets that are not represented in any of the dietary categories included in T-REX. Slightly more than half the species have diets that include large contributions by dietary items not included in T-REX. An analysis of risk using only T-REX based on simplistic diets is not adequate for a comprehensive assessment of risk from pesticides for all T&E bird species.

Rapid creation of stable mammalian cell lines for regulated expression of proteins using the Gateway® recombination cloning technology and Flp-In T-REx® lines.

The biochemical analysis of cellular processes in mammalian cells is often facilitated by the creation of cell lines coexpressing or overexpressing an affinity-tagged wild-type or mutant protein of interest in an inducible or noninducible stable manner (Malik and Roeder, 2003). The affinity tag allows for standardization of purification protocols to characterize interacting proteins or nucleic acids and minimizes the need for generating protein-specific antibodies at the early stages of analysis (for more information on affinity tags, see Purification of His-tagged proteins, Affinity purification of a recombinant protein expressed as a fusion with the maltose-binding protein (MBP) tag, Purification of GST-tagged proteins, Protein Affinity Purification using Intein/Chitin Binding Protein Tags, Immunoaffinity purification of proteins or Strep-tagged protein purification). The establishment of stable cell lines with inducible expression is critical to studying proteins that reduce cell growth and/or viability upon overexpression. Over the past several years, our laboratory has developed an expression platform for analyzing RNA-interacting proteins, including the establishment of stable mammalian cell lines expressing proteins of interest using a recombination-based cloning technology (Landthaler et al., 2008; Hafner et al., 2010). Our aim is to determine the mRNA targets of the hundreds of RNA-binding proteins encoded in the human genome by the isolation and molecular characterization of their ribonucleoprotein complexes (RNPs).

Aparelho T-Hyrax: um bem necessário / T-Hyrax appliance: a necessary change

Os aparelhos dentomucossuportados fazem parte do arsenal ortodôntico e/ou ortopédico para o tratamento da atresia maxilar (Haas) e podem estar associados às molas de TMA para correção da relação molar de Classe II por meio de distalização dos dentes superiores, sem depender da cooperação do paciente (Pêndulo, Pendex, T-Rex). Esses aparelhos têm a sua higiene dificultada e, ocasionalmente, promovem lesões à mucosa palatina. O presente artigo tem como objetivo expor uma discussão concernente às intercorrências clínicas relacionadas com a utilização de aparelhos que exibem apoio mucoso e apresentar uma associação do T-Rex ao Hyrax — o aparelho T-Hyrax — como uma alternativa para os casos que requerem a expansão rápida da maxila e a distalização de molares superiores, viabilizando, devido ao arquétipo do aparelho, uma condição de higiene mais favorável e com menores riscos de danos ao palato.