T4 Mutants Research Articles

Manipulating Interactions between T4 Phage Long Tail Fibers and Escherichia coli Receptors.

Bacteriophages are the most abundant and diverse biological entities on Earth. Phages exhibit strict host specificity that is largely conferred by adsorption. However, the mechanism underlying this phage host specificity remains poorly understood. In this study, we examined the interaction between outer membrane protein C (OmpC), one of the Escherichia coli receptors, and the long tail fibers of bacteriophage T4. T4 phage uses OmpC of the K-12 strain, but not of the O157 strain, for adsorption, even though OmpCs from the two E. coli strains share 94% homology. We identified amino acids P177 and F182 in loop 4 of the K-12 OmpC as essential for T4 phage adsorption in the copresence of loops 1 and 5. Analyses of phage mutants capable of adsorbing to OmpC mutants demonstrated that amino acids at positions 937 and 942 of the gp37 protein, which is present in the distal tip (DT) region of the T4 long tail fibers, play an important role in adsorption. Furthermore, we created a T4 phage mutant library with artificial modifications in the DT region and isolated and characterized multiple phage mutants capable of adsorbing to OmpC of the O157 strain or lipopolysaccharide of the K-12 strain. These results shed light on the mechanism underlying the phage host specificity mediated by gp37 and OmpC and may be useful in the development of phage therapy via artificial modifications of the DT region of T4 phage. IMPORTANCE Understanding the host specificity of phages will lead to the development of phage therapy. The interaction between outer membrane protein C (OmpC), one of the Escherichia coli receptors, and the gp37 protein present in the distal tip (DT) region of the long tail fibers of T4 bacteriophages largely determines their host specificity. Here, we elucidated the amino acid residues important for the interaction between gp37 and OmpC. This result suggests that the shapes of both proteins at the binding interface play important roles in their interactions, which are likely mediated by multiple residues of both binding partners. Additionally, we successfully isolated multiple phage mutants capable of adsorbing to a variety of E. coli receptors using a mutant T4 phage library with artificial modifications in the DT region, providing a foundation for the alteration of the host specificity.

Mouse model suggests limited role for human mesotrypsin in pancreatitis

Mesotrypsin is a low-abundance human trypsin isoform with a unique evolutionary mutation that conferred resistance to trypsin inhibitors and restricted substrate specificity. Mesotrypsin degrades the serine protease inhibitor Kazal type 1 (SPINK1) and thereby might increase risk for pancreatitis. Here, we report a mouse model designed to test the role of mesotrypsin in pancreatitis. We introduced the human mesotrypsin evolutionary signature mutation into mouse cationic trypsinogen (isoform T7), resulting in a Gly to Arg change at the corresponding position 199. In biochemical experiments using purified proteins, the p.G199R T7 mutant recapitulated all salient features of human mesotrypsin. T7G199R mice developed normally with no spontaneous pancreatitis or other obvious phenotypic changes. Cerulein-induced acute pancreatitis in C57BL/6N and T7G199R mice showed similar severity with respect to inflammatory parameters and acinar cell necrosis while plasma amylase activity was higher in T7G199R mice. Neither SPINK1 degradation nor elevated intrapancreatic trypsin activation was apparent in T7G199R mice. The results indicate that in T7G199R mice the newly created mesotrypsin-like activity has no significant impact on cerulein-induced pancreatitis. The observations suggest that human mesotrypsin is unimportant for pancreatitis; a notion that is consistent with published human genetic studies.

Fine-tuning sugar content in strawberry

Fine-tuning quantitative traits for continuous subtle phenotypes is highly advantageous. We engineer the highly conserved upstream open reading frame (uORF) of FvebZIPs1.1 in strawberry (Fragaria vesca), using base editor A3A-PBE. Seven novel alleles are generated. Sugar content of the homozygous T1 mutant lines is 33.9–83.6% higher than that of the wild-type. We also recover a series of transgene-free mutants with 35 novel genotypes containing a continuum of sugar content. All the novel genotypes could be immediately fixed in subsequent generations by asexual reproduction. Genome editing coupled with asexual reproduction offers tremendous opportunities for quantitative trait improvement.

Novel Escherichia coli RNA Polymerase Binding Protein Encoded by Bacteriophage T5.

The Escherichia coli bacteriophage T5 has three temporal classes of genes (pre-early, early, and late). All three classes are transcribed by host RNA polymerase (RNAP) containing the σ70 promoter specificity subunit. Molecular mechanisms responsible for the switching of viral transcription from one class to another remain unknown. Here, we find the product of T5 gene 026 (gpT5.026) in RNAP preparations purified from T5-infected cells and demonstrate in vitro its tight binding to E. coli RNAP. While proteins homologous to gpT5.026 are encoded by all T5-related phages, no similarities to proteins with known functions can be detected. GpT5.026 binds to two regions of the RNAP β subunit and moderately inhibits RNAP interaction with the discriminator region of σ70-dependent promoters. A T5 mutant with disrupted gene 026 is viable, but the host cell lysis phase is prolongated and fewer virus particles are produced. During the mutant phage infection, the number of early transcripts increases, whereas the number of late transcripts decreases. We propose that gpT5.026 is part of the regulatory cascade that orchestrates a switch from early to late bacteriophage T5 transcription.

Loss of function of CRT1a (calreticulin) reduces plant susceptibility toVerticillium longisporumin bothArabidopsis thalianaand oilseed rape (Brassica napus)

Summary Brassica napus is highly susceptible towards Verticillium longisporum (Vl43) with no effective genetic resistance. It is believed that the fungus reprogrammes plant physiological processes by up‐regulation of so‐called susceptibility factors to establish a compatible interaction. By transcriptome analysis, we identified genes, which were activated/up‐regulated in rapeseed after Vl43 infection. To test whether one of these genes is functionally involved in the infection process and loss of function would lead to decreased susceptibility, we firstly challenged KO lines of corresponding Arabidopsis orthologs with Vl43 and compared them with wild‐type plants. Here, we report that the KO of AtCRT1a results in drastically reduced susceptibility of plants to Vl43. To prove crt1a mutation also decreases susceptibility in B. napus, we identified 10 mutations in a TILLING population. Three T3 mutants displayed increased resistance as compared to the wild type. To validate the results, we generated CRISPR/Cas‐induced BnCRT1a mutants, challenged T2 plants with Vl43 and observed an overall reduced susceptibility in 3 out of 4 independent lines. Genotyping by allele‐specific sequencing suggests a major effect of mutations in the CRT1a A‐genome copy, while the C‐genome copy appears to have no significant impact on plant susceptibility when challenged with Vl43. As revealed by transcript analysis, the loss of function of CRT1a results in activation of the ethylene signalling pathway, which may contribute to reduced susceptibility. Furthermore, this study demonstrates a novel strategy with great potential to improve plant disease resistance.

Targeted mutagenesis in ryegrass (Lolium spp.) using the CRISPR/Cas9 system.

Targeted mutagenesis in ryegrass (Lolium spp.) using the CRISPR/Cas9 system.

Generation of seed lipoxygenase-free soybean using CRISPR-Cas9

Beany flavor induced by three lipoxygenases (LOXs, including LOX1, LOX2, and LOX3) restricts human consumption of soybean. It is desirable to generate lipoxygenase-free new mutant lines to improve the eating quality of soybean oil and protein products. In this study, a pooled clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) strategy targeting three GmLox genes (GmLox1, GmLox2, and GmLox3) was applied and 60 T0 positive transgenic plants were generated, carrying combinations of sgRNAs and mutations. Among them, GmLox-28 and GmLox-60 were gmlox1gmlox2gmlox3 triple mutants and GmLox-40 was a gmlox1gmlox2 double mutant. Sequencing of T1 mutant plants derived from GmLox-28, GmLox-60, and GmLox-40 showed that mutation in the GmLox gene was inherited by the next generation. Colorimetric assay revealed that plants carrying different combinations of mutations lost the corresponding lipoxygenase activities. Transgene-free mutants were obtained by screening the T2 generation of lipoxygenase-free mutant lines (GmLox-28 and GmLox-60). These transgene- and lipoxygenase-free mutants could be used for soybean beany flavor reduction without restriction by regulatory frameworks governing transgenic organisms.

Impact of bacteria motility in the encounter rates with bacteriophage in mucus

Bacteriophages—or phages—are viruses that infect bacteria and are present in large concentrations in the mucosa that cover the internal organs of animals. Immunoglobulin (Ig) domains on the phage surface interact with mucin molecules, and this has been attributed to an increase in the encounter rates of phage with bacteria in mucus. However, the physical mechanism behind this phenomenon remains unclear. A continuous time random walk (CTRW) model simulating the diffusion due to mucin-T4 phage interactions was developed and calibrated to empirical data. A Langevin stochastic method for Escherichia coli (E. coli) run-and-tumble motility was combined with the phage CTRW model to describe phage-bacteria encounter rates in mucus for different mucus concentrations. Contrary to previous theoretical analyses, the emergent subdiffusion of T4 in mucus did not enhance the encounter rate of T4 against bacteria. Instead, for static E. coli, the diffusive T4 mutant lacking Ig domains outperformed the subdiffusive T4 wild type. E. coli’s motility dominated the encounter rates with both phage types in mucus. It is proposed, that the local fluid-flow generated by E. coli’s motility combined with T4 interacting with mucins may be the mechanism for increasing the encounter rates between the T4 phage and E. coli bacteria.

Aklimatisasi dan Evaluasi Produksi Mutan Rumput Gajah Kultivar Taiwan

Cultivation of the Elephant Grass cv Taiwan with tissue culture requires an acclimatization stage, therefore this study aims to find the best planting media and the effect of plant age and evaluation of production elephant grass cv taiwan mutant and nutrient content. Acclimatization was carried out in a greenhouse using sand, soil, and a combination of 50% sand with 50% soil, and the mutant type T2, T4 and K4. The acclimation process uses the Factorial RAL method with factor A type of mutant and factor B type of planting media and the parameters assessed are leaf length, number of leaves and plant mortality rate. Production evaluation uses the RAL method and the parameters observed to see elephant grass production are plant height, stem diameter, leaf width, number of tillers and amount of plant biomass production. Proximate analysis is used to show the nutritional value of plants that have been selected individually using scoring assessments. The results of the acclimatization study showed that the planting media that had the lowest mortality rate were sand and soil combination media (22%) followed by soil planting media (27%) and the planting media that had the highest mortality rate was sand media (30%). The results of the production evaluation stage showed that the highest average harvest weight was found in T4 mutants with an average weight of 1353 g clump-1harvest-1, followed by T2 mutants which had an average of 1198 g clump-1harvest-1 and the lowest harvest weight was found in K4 mutants with an average of 1002 g clump-1harvest-1. Based on the research results obtained the best planting media for the acclimation process of elephant grass cv Taiwan mutants is a combination of sand and soil media (1:1). The results of the evaluation stage of T4 mutant production are the best mutants based on morphological performance and production of plant biomass. Proximate analysis of selected plants shows that plants with mutant T2PT7 species have the best nutritional value, with nutritional content of PK 11.71 and SK 24.67.

NSs Filament Formation Is Important but Not Sufficient for RVFV Virulence In Vivo.

Rift Valley fever virus (RVFV) is a mosquito-borne phlebovirus that represents as a serious health threat to both domestic animals and humans. The viral protein NSs is the key virulence factor of RVFV, and has been proposed that NSs nuclear filament formation is critical for its virulence. However, the detailed mechanisms are currently unclear. Here, we generated a T7 RNA polymerase-driven RVFV reverse genetics system based on a strain imported into China (BJ01). Several NSs mutations (T1, T3 and T4) were introduced into the system for investigating the correlation between NSs filament formation and virulence in vivo. The NSs T1 mutant showed distinct NSs filament in the nuclei of infected cells, the T3 mutant diffusively localized in the cytoplasm and the T4 mutant showed fragmented nuclear filament formation. Infection of BALB/c mice with these NSs mutant viruses revealed that the in vivo virulence was severely compromised for all three NSs mutants, including the T1 mutant. This suggests that NSs filament formation is not directly correlated with RVFV virulence in vivo. Results from this study not only shed new light on the virulence mechanism of RVFV NSs but also provided tools for future in-depth investigations of RVFV pathogenesis and anti-RVFV drug screening.

Changes of Thermostability, Organic Solvent, and pH Stability in Geobacillus zalihae HT1 and Its Mutant by Calcium Ion.

Thermostable T1 lipase from Geobacillus zalihae has been crystallized using counter-diffusion method under space and Earth conditions. The comparison of the three-dimensional structures from both crystallized proteins show differences in the formation of hydrogen bond and ion interactions. Hydrogen bond and ion interaction are important in the stabilization of protein structure towards extreme temperature and organic solvents. In this study, the differences of hydrogen bond interactions at position Asp43, Thr118, Glu250, and Asn304 and ion interaction at position Glu226 was chosen to imitate space-grown crystal structure, and the impact of these combined interactions in T1 lipase-mutated structure was studied. Using space-grown T1 lipase structure as a reference, subsequent simultaneous mutation D43E, T118N, E226D, E250L, and N304E was performed on recombinant wild-type T1 lipase (wt-HT1) to generate a quintuple mutant term as 5M mutant lipase. This mutant lipase shared similar characteristics to its wild-type in terms of optimal pH and temperature. The stability of mutant 5M lipase improved significantly in acidic and alkaline pH as compared to wt-HT1. 5M lipase was highly stable in organic solvents such as dimethyl sulfoxide (DMSO), methanol, and n-hexane compared to wt-HT1. Both wild-type and mutant lipases were found highly activated in calcium as compared to other metal ions due to the presence of calcium-binding site for thermostability. The presence of calcium prolonged the half-life of mutant 5M and wt-HT1, and at the same time increased their melting temperature (Tm). The melting temperature of 5M and wt-HT1 lipases increased at 8.4 and 12.1 °C, respectively, in the presence of calcium as compared to those without. Calcium enhanced the stability of mutant 5M in 25% (v/v) DMSO, n-hexane, and n-heptane. The lipase activity of wt-HT1 also increased in 25% (v/v) ethanol, methanol, acetonitrile, n-hexane, and n-heptane in the presence of calcium. The current study showed that the accumulation of amino acid substitutions D43E, T118N, E226D, E250L, and N304E produced highly stable T1 mutant when hydrolyzing oil in selected organic solvents such as DMSO, n-hexane, and n-heptane. It is also believed that calcium ion plays important role in regulating lipase thermostability.

Genome sequencing and CRISPR/Cas9 gene editing of an early flowering Mini-Citrus (Fortunella hindsii).

SummaryHongkong kumquat (Fortunella hindsii) is a wild citrus species characterized by dwarf plant height and early flowering. Here, we identified the monoembryonic F. hindsii (designated as ‘Mini‐Citrus’) for the first time and constructed its selfing lines. This germplasm constitutes an ideal model for the genetic and functional genomics studies of citrus, which have been severely hindered by the long juvenility and inherent apomixes of citrus. F. hindsii showed a very short juvenile period (~8 months) and stable monoembryonic phenotype under cultivation. We report the first de novo assembled 373.6 Mb genome sequences (Contig‐N50 2.2 Mb and Scaffold‐N50 5.2 Mb) for F. hindsii. In total, 32 257 protein‐coding genes were annotated, 96.9% of which had homologues in other eight Citrinae species. The phylogenomic analysis revealed a close relationship of F. hindsii with cultivated citrus varieties, especially with mandarin. Furthermore, the CRISPR/Cas9 system was demonstrated to be an efficient strategy to generate target mutagenesis on F. hindsii. The modifications of target genes in the CRISPR‐modified F. hindsii were predominantly 1‐bp insertions or small deletions. This genetic transformation system based on F. hindsii could shorten the whole process from explant to T1 mutant to about 15 months. Overall, due to its short juvenility, monoembryony, close genetic background to cultivated citrus and applicability of CRISPR, F. hindsii shows unprecedented potentials to be used as a model species for citrus research.

Building an Inducible T7 RNA Polymerase/T7 Promoter Circuit in Synechocystis sp. PCC6803.

To develop tightly regulated orthogonal gene expression circuits in the photoautotrophic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we designed a circuit in which a native inducible promoter drives the expression of phage T7 RNA polymerase (T7RNAP). T7RNAP, in turn, specifically recognizes the T7 promoter that is designed to drive GFP expression. In Syn6803, this T7RNAP/T7promoter-GFP circuit produces high GFP fluorescence, which was further enhanced by using mutant T7 promoters. We also tested two orthogonal inducible promoters, Trc1O and L03, but these promoters drive T7RNAP to levels that are toxic in E. coli. Introduction of a protein degradation tag alleviated this problem. However, in Syn6803, these circuits did not function successfully. This highlights the underappreciated fact that similar circuits work with varying efficiencies in different chassis organisms. This lays the groundwork for developing new orthogonally controlled phage RNA polymerase-dependent expression systems in Syn6803.

Mutant T4 DNA polymerase for easy cloning and mutagenesis.

The advent of high-fidelity DNA polymerases that can be used to linearize and amplify whole plasmids by PCR opened the door to greatly simplified cloning and mutagenesis protocols. Commercially available kits work well, but often have been optimized using undisclosed or proprietory components. Here we show that a mutant T4 DNA polymerase (Y320A) with attenuated 3’-exonuclease activity is uniquely suited to generate single-stranded DNA overhangs of uniform length in a more easily controllable manner than the wild-type enzyme, and this can be used to increase the yields of colonies containing correctly modified plasmids in cloning and mutagenesis experiments, which is particularly useful when E. coli cells are of relatively low competency. Standard protocols using the mutant T4 DNA polymerase are provided for the sequence and ligation independent cloning (SLIC) method and a modified QuikChange method, where the mutant enzyme enhances the yield of correctly mutated plasmid and further suppresses parental plasmid during digestion with DpnI. Single-stranded DNA overhangs generated by the mutant T4 DNA polymerase facilitate subsequent plasmid circularization, annealing and ligation in E. coli.

Absence of Calreticulin Phenocopies Cellular Abnormalities Induced By Calreticulin Exon-9 Mutation in Myeloproliferative Neoplasms

INTRODUCTION. Calreticulin (CALR) is mutated in 20% of pts with essential thrombocythemia (ET) and primary myelofibrosis (PMF). The most frequent mutations are a 52 bp deletion (T1) and a 5 bp insertion (T2) in exon 9, that cause a recurrent frameshift resulting in novel C-terminal sequence common to all mutant CALR proteins. Recent data indicate that interaction of mutant CALR with the thrombopoietin receptor MPL contributes to the abnormal megakaryocytopoiesis (Mk) of ET/PMF (Araki M et al & Chachoua I et al, Blood 2016; Elif S et al, Blood 2018), however full characterization of mechanisms pertaining to mutant CALR remains to be pursued.METHODS. By using CRISPR/Cas9 editing, we generated CALR knock-out (KO) variants starting from cord blood (CB) CD34+ cells, K562, UT7 and HL-60 cell lines, and CALR T1 variants from K562 and UT7 cells. Stable expression of CALR wild-type (WT), T1 and T2 was obtained by viral transfection of K562-KO cells.RESULTS. In the different genome-edited cell models, KO or T1 mutation did not result in appreciable changes in proliferation rate, cell cycle and apoptosis under standard culture conditions. However, UT7 KO and T1 cells were able to grow in the absence of GM-CSF, that was otherwise necessary for maintenance of WT counterpart, indicating cytokine independence similarly imparted by deletion or T1 mutation of CALR. To evaluate impact of mutated CALR on Mk commitment, we induced parental, KO and T1 K562 cells with phorbol-myristate acetate (PMA); at day 3 after induction, 60% and 48% of KO and T1 cells, respectively, expressed CD41/CD61 compared to 24% of parental cells (p<0.01). Similar findings were noted in KO K562 cells that were transfected with vectors encoding the WT, T1 and T2 mutated CALR: CD41/CD61+ cells increased from 30% in parental cells to 70%, 55%, 62%, respectively, in KO cells and cells expressing T1 and T2 mutant CALR (p<0.01). We also found that a greater proportion of KO and T1 UT7 cells, maintained in either GM-CSF and TPO, spontaneously expressed CD41/CD61 at d7 of culture (50-51% and 41-49%, respectively, for KO and T1 cells with GM-CSF or TPO compared to 26-25% of parental cells; p<0.01) and underwent morphologically recognizable Mk maturation. Mk clonogenic assays were initiated in plasma clot cultures by plating CB-derived CD34+ cells with CRISPR/Cas9-induced deletion of CALR (KO cells). KO CD34+ cells generated a number of CFU-Mk colonies that was 10-fold higher than untouched CD34+ cells (p<0.05). Overall, these data suggested that deletion of CALR results in promotion of Mk differentiation in cell lines and primary cells thereby mimicking the effects of CALR T1. Array phosphoproteomic assay in K562 KO cells showed phosphorylation levels of p38γ, p70S6k mTOR, Erk1 and Erk2 from 0.5 to >2-fold higher compared to parental cells. Then, we induced KO and T1 K562 cells to Mk differentiation in the presence of JAK2, mTOR and Erk1/2 -inhibitors; only mTOR and Erk1/2 -inhibitors significantly (P< 0.05) reduced Mk differentiation of T1 and KO cells by respectively 5- and 4- fold with mTOR inhibitor and 5- and 4.5- fold with Erk1/2 inhibitor compared to parental cells, that were largely insensitive to inhibitors (Figure).Isolated megakaryocytes from CALR-mutant patients were found to present abnormally increased release of Ca2+ from endoplasmic reticulum (ER) compared to control cells (Pietra D et al. Leukemia, 2016). We similarly found that both KO and T1 K562 cells presented impaired capability in restoring calcium homeostasis compared to parental cells under treatment with ionophores thapsigargin and ionomycin. Finally, we comparatively assessed MPO expression by flow cytometric analysis in parental and CALR KO HL-60 cell lines; increased myeloperoxidase (MPO) degradation was previously reported in MPN pts expressing CALR mutation (Theocharides A et al., Blood, 2016). We found significant reduction of MPO levels (9-fold lower, p<0.01) in KO compared to parental cells, while MPO mRNA levels were not affected by CALR KO, suggesting similarly enhanced degradation of MPO in cells with targeted deletion of CALR.CONCLUSIONS. Overall, our data suggest that the novel C-terminus of CALR originating from exon 9 mutations loses some physiologically functions that are phenocopied by absence of CALR protein in cells made KO for the gene in a MPL-independent context. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mutational Mtc6p attenuates autophagy and improves secretory expression of heterologous proteins in Kluyveromyces marxianus

BackgroundThe yeast Kluyveromyces marxianus is an emerging cell factory for heterologous protein biosynthesis and its use holds tremendous advantages for multiple applications. However, which genes influence the productivity of desired proteins in K. marxianus has so far been investigated by very few studies.ResultsIn this study, we constructed a K. marxianus recombinant (FIM1/Est1E), which expressed the heterologous ruminal feruloyl esterase Est1E as reporter. UV-60Co-γ irradiation mutagenesis was performed on this recombinant, and one mutant (be termed as T1) was screened and reported, in which the productivity of heterologous Est1E was increased by at least tenfold compared to the parental FIM1/Est1E recombinant. Transcriptional perturbance was profiled and presented that the intracellular vesicle trafficking was enhanced while autophagy be weakened in the T1 mutant. Moreover, whole-genome sequencing combined with CRISPR/Cas9 mediated gene-editing identified a novel functional protein Mtc6p, which was prematurely terminated at Tyr251 by deletion of a single cytosine at 755 loci of its ORF in the T1 mutant. We found that deleting C755 of MTC6 in FIM1 led to 4.86-fold increase in the production of Est1E compared to FIM1, while the autophagy level decreased by 47%; on the contrary, when reinstating C755 of MTC6 in the T1 mutant, the production of Est1E decreased by 66% compared to T1, while the autophagy level increased by 124%. Additionally, in the recombinant with attenuated autophagy (i.e., FIM1 mtc6C755Δ and T1) or interdicted autophagy (i.e., FIM1 atg1Δ and T1 atg1Δ), the productivity of three other heterologous proteins was also increased, specifically the heterologous mannase Man330, the β-1,4-endoxylanase XynCDBFV or the conventional EGFP.ConclusionsOur results demonstrated that Mtc6p was involved in regulating autophagy; attenuating or interdicting autophagy would dramatically improve the yields of desired proteins in K. marxianus, and this modulation could be achieved by focusing on the premature mutation of Mtc6p target.

Abstract 3925: Inhibition of oncogenic and drug-resistant PDGFRA and KIT alterations by DCC-2618

Abstract Introduction: Activating mutations and other genetic alterations in KIT and PDGFRA receptor tyrosine kinases have been identified in certain cancers and proliferative diseases, including most cases of gastrointestinal stromal tumors (GIST) and systemic mastocytosis, and small percentages of gliomas, lung cancer, and leukemias. The treatment of metastatic GIST has been transformed with KIT inhibitors, but heterogeneous drug-resistant mutations arise during therapy, with individual patients often having multiple KIT mutations in different tumor sites. PDGFRA variants in GIST and other cancers also have a significant unmet medical need. DCC-2618 is a kinase switch control inhibitor that potently inhibits the spectrum of exon 9, 11, 13, 14, 17 and 18 mutations in KIT and exons 12, 14 and 18 mutations in PDGFRA. DCC-2618 has been designed to bind as a type II kinase inhibitor that forces the mutant kinases, including strongly activated mutants such as D816V KIT and D842V PDGFRA, into inactive conformations. DCC-2618 has been observed to be potent in enzyme and cell-based assays, and has demonstrated consistent efficacy in xenograft models driven by PDGFRA and KIT alterations. Methods: DCC-2618, and an active human metabolite, DP-5439, were tested for inhibition of PDGFRA and KIT mutants using standard enzyme and binding assays, and a variety of cell-based assays. Levels of phosphorylated PDGFRA and KIT were determined by Western blot or ELISA. Proliferation was measured using the fluorescent dye resazurin. An x-ray crystal structure of an analog of DCC-2618 was determined at Emerald Biostructures. The H1703 PDGFRA-amplified lung cancer and GIST T1 mutant KIT xenograft models were performed at MI Bioresearch. A GIST PDX exon 17 mutant KIT xenograft model was run at Molecular Response. Results: DCC-2618 and the metabolite DP-5439 inhibited KIT and PDGFRA variants with nanomolar potency. In CHO cells transfected with KIT or PDGFRA variants, DCC-2618 was shown to inhibit the full spectrum of the clinically relevant primary and refractory drug-resistant mutations tested. DCC-2618 also inhibited phosphorylation of KIT or PDGFRA in cell lines with various drug-resistant KIT mutations or PDGFRA alterations. DCC-2618 was compared to the FDA-approved KIT inhibitors imatinib, sunitinib, regorafenib, and midostaurin, as well as other KIT and PDGFRA inhibitors. In vivo, treatment with DCC-2618 led to tumor regressions in KIT- and PDGFRA-driven xenograft models. Conclusions: DCC-2618 has been observed to be a potent inhibitor of KIT and PDGFRA alterations, including mutants, fusions, and amplifications. Based on this profile, DCC-2618 may have utility in the treatment of KIT and PDGFRA-driven cancers including GIST, systemic mastocytosis, and a subset of lung cancers, gliomas, and leukemias. DCC-2618 is currently in a Phase 1 clinical trial in KIT and PDGFRA driven cancers (ClinicalTrials.gov Identifier: NCT02571036). Citation Format: Bryan D. Smith, Michael D. Kaufman, Anu Gupta, Cynthia B. Leary, Wei-ping Lu, Stacie L. Bulfer, Gada Al-Ani, Jarnail Singh, Subha Vogeti, Michael C. Heinrich, Daniel L. Flynn. Inhibition of oncogenic and drug-resistant PDGFRA and KIT alterations by DCC-2618 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3925.

Biocontrol potential of saline- or alkaline-tolerant Trichoderma asperellum mutants against three pathogenic fungi under saline or alkaline stress conditions.

Salinity and alkalinity are major abiotic stresses that limit growth and development of poplar. We investigated biocontrol potential of saline- and alkaline-tolerant mutants of Trichoderma asperellum to mediate the effects of salinity or alkalinity stresses on Populus davidiana × P. alba var. pyramidalis (PdPap poplar) seedlings. A T-DNA insertion mutant library of T. asperellum was constructed using an Agrobacterium tumefaciens mediated transformation system; this process yielded sixty five positive transformants (T1–T65). The salinity tolerant mutant, T59, grew in Potato Dextrose Agar (PDA) containing up to 10% (1709.40 mM) NaCl. Under NaCl-rich conditions, T59 was most effective in inhibiting Alternaria alternata (52.00%). The alkalinity tolerant mutants, T3 and T5, grew in PDA containing up to 0.4% (47.62 mM) NaHCO3. The ability of the T3 and T5 mutants to inhibit Fusarium oxysporum declined as NaHCO3 concentrations increased. NaHCO3 tolerance of the PdPap seedlings improved following treatment with the spores of the WT, T3, and T5 strains. The salinity tolerant mutant (T59) and two alkalinity tolerant mutants (T3 and T5) generated in this study can be applied to decrease the incidence of pathogenic fungi infection under saline or alkaline stress.

Construction of synthetic T7 RNA polymerase expression systems.

T7 RNA polymerase (T7 RNAP) is one of the preferred workhorses for recombinant gene expression, owing in part to its high transcriptional activity and the fact that it has a small (17 base-pair), easily manipulated promoter. Furthermore, the fact that T7 RNAP is largely orthogonal to most hosts enables its use in a wide variety of contexts. However, the high activity of the enzyme also often leads to an increased fitness burden on the host, limiting the predictability of its interactions and impact on host physiology, and potentially leading to mutations in the constructs. Here we use a synthetic biology approach to design and characterize a panel of T7 RNAP expression circuits with different modes of regulation that enable the reliable expression of downstream targets under a variety of conditions. First, we describe the construction of a minimal T7 RNAP expression system that is inducible by a small molecule anhydrotetracycline (aTc), and then characterize a self-limiting T7 RNAP expression circuit that provides better control over the amount of T7 RNAP produced upon induction. Finally, we characterize a so-called T7 RNAP homeostasis circuit that leads to constitutive, continuous, and sub-toxic levels of T7 RNAP. Coupled with previously characterized mutant T7 RNAP promoters in vitro, we demonstrate that this modular framework can be used to achieve precise and predictable levels of output (sfGFP) in vivo. This new framework should now allow modeling and construction of T7 RNAP expression constructs that expand the utility of this enzyme for driving a variety of synthetic circuits and constructs.

Search for independent (β/α)4 subdomains in a (β/α)8 barrel β-glucosidase.

Proteins that fold as (β/α)8 barrels are thought to have evolved from half-barrels that underwent duplication and fusion events. The evidence is particularly clear for small barrels, which have almost identical halves. Additionally, computational calculations of the thermodynamic stability of these structures in the presence of denaturants have revealed that (β/α)8 barrels contain two subunits or domains corresponding to half-barrels. Hence, within (β/α)8 barrels, half-barrels are self-contained units. Here, we tested this hypothesis using β-glucosidase from the bacterium Thermotoga maritima (bglTm), which has a (β/α)8 barrel structure. Mutations were introduced to disrupt the noncovalent contacts between its halves and reveal the presence of two domains within bglTm, thus resulting in the creation of mutants T1 (containing W12A and I217A mutations) and T2 (containing W12A, H195A, I217A and F404A mutations). Mutants T1 and T2 were properly folded, as indicated by their fluorescence spectra and enzyme kinetic parameters. T1 and wild-type bglTm were equally stable, as shown by the results of thermal inactivation, differential scanning fluorimetry and guanidine hydrochloride denaturation experiments. However, T2 showed a first-order inactivation at 80°C, a single melting temperature of 82°C and only one transition concentration (c50) in 2.4 M guanidine hydrochloride. Additionally, T1 and T2 exhibited a cooperative denaturation process that followed a two-state model (m-values equal to 1.4 and 1.6 kcal/mol/M, respectively), similar to that of wild-type bglTm (1.2 kcal/mol/M). Hence, T1 and T2 each denatured as a single unit, although they contained different degrees of disruption between their halves. In conclusion, bglTm halves are equivalent in terms of their thermal and chemical stability; thus, their separate contributions to (β/α)8 barrel unfolding cannot be disentangled.