Spatio-temporal control of phenylpropanoid biosynthesis by inducible complementation of a cinnamate 4-hydroxylase mutant

Cinnamate 4-hydroxylase (C4H) is a cytochrome P450-dependent monooxygenase that catalyzes the second step of the general phenylpropanoid pathway. Arabidopsis reduced epidermal fluorescence 3 (ref3) mutants, which carry hypomorphic mutations in C4H, exhibit global alterations in phenylpropanoid biosynthesis and have developmental abnormalities including dwarfing. Here we report the characterization of a conditional Arabidopsis C4H line (ref3-2 pOpC4H), in which wild-type C4H is expressed in the ref3-2 background. Expression of C4H in plants with well-developed primary inflorescence stems resulted in restoration of fertility and the production of substantial amounts of lignin, revealing that the developmental window for lignification is remarkably plastic. Following induction of C4H expression in ref3-2 pOpC4H, we observed rapid and significant reductions in the levels of numerous metabolites, including several benzoyl and cinnamoyl esters and amino acid conjugates. These atypical conjugates were quickly replaced with their sinapoylated equivalents, suggesting that phenolic esters are subjected to substantial amounts of turnover in wild-type plants. Furthermore, using localized application of dexamethasone to ref3-2 pOpC4H, we show that phenylpropanoids are not transported appreciably from their site of synthesis. Finally, we identified a defective Casparian strip diffusion barrier in the ref3-2 mutant root endodermis, which is restored by induction of C4H expression.

Can Turn-Taking Highlight the Nature of Non-Verbal Behavior: A Case Study

The present research explores non-verbal behavior that accompanies the management of turns in naturally occurring conversations. To analyze turn management, we implemented the ISO 24617-2 multidimensional dialog act annotation scheme. The classification of the communicative intent of non-verbal behavior was performed with the annotation scheme for spontaneous authentic communication called the EVA annotation scheme. Both dialog acts and non-verbal communicative intent were observed according to their underlying nature and information exchange channel. Both concepts were divided into foreground and background expressions. We hypothesize that turn management dialog acts, being a background expression, co-occur with communication regulators, a class of non-verbal communicative intent, which are also of background nature. Our case analysis confirms this hypothesis. Furthermore, it reveals that another group of non-verbal communicative intent, the deictics, also often accompany turn management dialog acts. As deictics can be both foreground and background expressions, the premise that background non-verbal communicative intent is interlinked with background dialog acts is upheld. And when deictics were perceived as part of the foreground they co-occurred with foreground dialog acts. Therefore, dialog acts and non-verbal communicative intent share the same underlying nature, which implies a duality of the two concepts.

A low-background Tet-On system based on post-transcriptional regulation using Csy4

On account of its stringent regulation and high rate of induction, the tetracycline regulatory system is used extensively for inducing target gene expression in eukaryotes. However, under certain circumstances, its associated background expression can be problematic, as in the expression of highly toxic proteins. We found that when using the Tet-On 3G system to drive expression of the kid toxin gene in sf9 insect cells, a higher percentage of cells were killed than when using an empty vector in the absence of the induction agent doxycycline, thereby indicating the leaky expression of this inducible expression system. Moreover, we found that the tetracycline-controlled transcriptional silencer (tTS) does not effectively reduce the background expression of the Tet-On 3G system in sf9 cells. However, Csy4, a Cas9 homologous protein in the CRISPR family with sequence-specific endonuclease activity, was found to be effective in reducing the Tet-On 3G system-associated background expression, although there was a concomitant reduction in the maximum induced expression. Nevertheless, we found that modification of the system via incorporation of TRE-controlled anti-sense csy4 in combination with a WSSVie1 (Δ23) promotor-driven sense csy4 significantly reduced the leaky expression of the Tet-On 3G system, and that the level of induction was higher than that initially obtained. This optimized Tet-On 3G system can significantly reduce cell death attributed to the background expression of Kid under uninduced conditions. Therefore, we developed a novel low-background inducible expression system for use in insect cells and potentially in other organisms including mammals based on post-transcriptional regulation using Csy4.

Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid

We previously described the design of triacetic acid lactone (TAL) biosensor “AraC-TAL1”, based on the AraC regulatory protein. While useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity, and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a “decoy” ligand during negative selection, resulting in isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the IPRO computational framework was employed, followed by two rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.

Evaluating the effects of synthetic POM cycles and NAD kinase expression on fatty alcohol production in Saccharomyces cerevisiae

Background: NADPH-dependent enzymes play important roles in many anabolic reactions and the availability of redox cofactors can influence metabolic flux ultimately influencing titers of bioproducts produced by engineered microbial cells. This may be especially true of oleochemical production when carbon flux through the highly NADPH-dependent fatty acid biosynthesis pathway is increased. While pathway specific approaches are often applied to counter redox imbalance, a study evaluating generalized approaches to improved NADPH availability is lacking in Saccharomyces cerevisiae . Results: Here, we have created four unique synthetic Pyruvate-Oxaloacetate-Malate “POM” cycles consisting of either of the endogenous isoforms of pyruvate carboxylase ( PYC1 or PYC2 ), a modified version of malate dehydrogenase ( ‘MDH1 or ‘MDH2 ), and a truncated cytosolic form of the endogenous malic enzyme ( sMAE1 ). Only the POM cycle that combined expression of PYC1 , ‘MDH2 , and sMAE1 increased the titer of fatty alcohols produced; however, it did so in two unique fatty alcohol producing strains. In a FAS1 overexpression background, expression of this synthetic POM cycle increased fatty alcohol titers by 40% from 49.0 ± 2.2 mg/L to 68.6 ± 3.3 mg/L and showed similar results in a zwf1 deletion strain. The effect of overexpression of the endogenous NAD+ kinases UTR1 , YEF1 , and a cytosolic version of POS5 were also tested. We found that expression of POS5c resulted in an ~35% increase in fatty alcohol titer, while the overexpression of the UTR1 or YEF1 did not significantly influence titers. In these minimally engineered cells, combined overexpression of PYC1 , ‘ MDH2 , sMAE1 and POS5c did not further increase titers Conclusions: Overexpression of PYC1 in conjunction with ‘MDH2 and sMAE1 results in a synthetic POM cycle which can be utilized to improve fatty alcohol production in engineered strains of S. cerevisiae . Additionally, overexpression of a truncated version of POS5 ( POS5c ) results in similar increases in fatty alcohol production. These findings may serve to provide a generalized mechanism to increase NADPH production in engineered cells, resulting in increased bioproduct titers.

Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid

We previously described the design of triacetic acid lactone (TAL) biosensor ‘AraC-TAL1’, based on the AraC regulatory protein. Although useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a ‘decoy’ ligand during negative selection, resulting in the isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the iterative protein redesign and optimization computational framework was employed, followed by 2 rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.

Effect of UBE2Q on BECN and CEA Protein in Colorectal Cancer Cells

Background: Expression of UBE2Q1, UBE2Q2, and members of the ubiquitinconjugating enzyme family (E2) are affected in colorectal cancer (CRC). The BECN gene plays a key role in CRC cells. In gastrointestinal carcinoma therapy, tumorassociated antigens such as CEA are typically used.To investigate the association between UBE2Q1 and Beclin1 autophagy marker and CEA protein expression in LS180 CRC cell line. Materials and Methods: In this study, changes in the expression of BECN marker in LS180 cell lines with the vector containing UBE2Q1 were investigated using real-time PCR. The expression of CEA protein was also evaluated by western blotting. Statistical analyses were performed with Graph Pad Prism software. Results: The results indicated reduced expression of BECN autophagy marker (P=0). Therefore, in the presence of UBE2Q1, cancer cells have less ability to induce autophagy. However, CEA protein levels in LS180 transfected cells with a UBE2Q1- ORF-containing plasmid decreased when compared to non-transfected cells. Conclusion: The use of pharmacologic inhibitors related to the autophagy mechanism can be a novel approach in cancer therapy.

Retinoblastoma (Rb) protein expression in triple-negative breast cancer.

1097 Background: Expression of Rb, the protein product of the RB tumor suppressor gene, is required for cyclin D kinase (CDK) 4/6 inhibition in luminal breast cancers. Triple negative breast cancers (TNBC) frequently exhibit Rb loss and are thus believed to be poor candidates for CDK4/6 inhibition. However, the features associated with Rb loss in TNBC are poorly-defined. We aimed to assess whether TNBC that express the androgen receptor (AR) and resemble a more luminal subtype retain Rb more often than other TNBC. Methods: To assess the frequency and correlates of Rb expression in TNBC, we stained tissue microarrays (TMAs) containing 180 Stage I-III TNBC, 71 with germline BRCA1 mutations ( BRCA1+) and 109 without BRCA1 mutation (sporadic), using a mouse monoclonal antibody to human Rb (clone G3-245, BD Biosciences). Assessment of tumor size, histologic type, grade, lymphovascular invasion and lymph node status were assessed on histologic review. The TMAs, containing three 0.6mm cores/tumor, had been previously stained for AR, CK5/6, CK14 and EGFR. Log-binomial regression was used to calculate risk ratios (RR). Results: Fifty percent of TNBC were Rb-positive (Rb+; ≥10% nuclei staining) of which 84.4% had > 50% nuclei staining. Among TNBC that were Rb-negative, 76.6% had < 1% nuclei staining for Rb. AR expression (≥10% nuclei staining) was more common in Rb+ than Rb-negative TNBC (16.7% vs 4.5%; p = 0.01). In addition, Rb expression was significantly more common among sporadic than BRCA1+ TNBC (59.6% vs 35.2%, p = 0.001). Compared with Rb-negative TNBC, Rb+ tumors were associated with older age (mean 48.7 vs 45.7 years; p = 0.06) and lower histologic grade (grade 1 or 2: 9.2% vs 2.2%; p = 0.05). No other clinical or pathologic features were significantly associated with Rb status. In a multivariable model, both AR expression (RR, 1.65; 95% CI, 1.31-2.08) and lack of BRCA1 mutation (RR, 1.63; 95% CI, 1.17-2.29) significantly predicted for Rb expression among TNBC. Conclusions: Sporadic TNBC is significantly more likely to be Rb+ than BRCA1+ TNBC. Among TNBC, AR expression significantly predicts for Rb expression. These results suggest clinically relevant biomarkers that may predict for Rb expression and potential targeted therapies in TNBC.

Genome-wide, CRISPR-Cas9-based genetic screening for regulators of interferon-induced PD-L1 expression in AML cells.

116 Background: Expression of PD-L1 in AML cells is induced by pro-inflammatory signals, but how the induction is regulated is incompletely known. Genome-wide screening using the CRISPR-Cas9 technology could provide a strategy to interrogate the regulators of PD-L1 expression. Methods: A lentiviral CRISPR-Cas9 library, containing 3 sgRNA targeting each of 19,050 human genes (Nat Methods 2014, 11:783), was used to transduce THP-1 AML cells. Transduced cells were treated with IFN-γ and stained with an anti-PD-L1 antibody. Two subsets of cells were isolated by cell sorting according to their levels of PD-L1 expression. One subset consisted of cells that showed the median 5% of PD-L1 expression and the other subset, the lowest 5%. sgRNA integrated in the sorted subsets were amplified and their frequencies quantified by deep sequencing. Results: As expected, sgRNA targeting the PD-L1 gene itself were highly enriched in the subset that expressed a low level of PD-L1. sgRNA that targeted IFNGR2, β-chain of the IFN-γ receptor, were also enriched in this subset, whereas those for IFNGR1 were not. sgRNA targeting both JAK1 and JAK2 were enriched. By contrast, those for JAK3 and TYK2 were not. Enrichment was seen for sgRNA that targeted STAT1, a transcriptional activator downstream of JAK1/2, whereas sgRNA targeting the other STAT activators (STAT-2, 3, 4, 5A, 5B and 6) showed no enrichment. sgRNA targeting several serine kinases that regulate STAT1 phosphorylation (PKCδ, PKCε, PKCα, CAMKII, ERK, JNK, and CDK8) were not enriched. Nor were sgRNA targeting several phosphatases that modulate STAT1 activity (TC-PTP, PTPN1, SHP1 and SHP2). sgRNA targeting IRF1 (IFN regulatory transcription factor 1) were highly enriched, whereas sgRNA for IRF-2 to IRF-9 were not. In each of the affected genes, at least 2 out of the 3 sgRNA targeting the gene were enriched, indicating a generality of the findings. Conclusions: A CRISPR-Cas9-based screening strategy provided an unbiased interrogation of genes essential for PD-L1 expression on AML cells. It confirmed the importance of the IFNGR2-JAK1/2-STAT1-IRF1 axis, which could serve as targets for the development of drugs to suppress the ability of AML cells to evade host immunity.