Combination Of Mutant Research Articles

Enhanced trans-cleavage activity using CRISPR-Cas12a variant designed to reduce steric inhibition by cis-cleavage products

The CRISPR-Cas12a system has emerged as a promising tool for molecular diagnostics due to its indiscriminate trans-ssDNase activity. However, the sensitivity of Cas12a-based diagnostics remains insufficient for clinical use without a pre-amplification step such as loop-mediated isothermal amplification, and therefore the trans-cleavage activity of Cas12a needs to be enhanced. Here, we present a novel strategy to enhance the trans-cleavage activity of Cas12a by reducing the steric hindrance from cis-cleavage products. We have designed Cas12a variants with alanine mutations in the target strand loading (TSL) domain, resulting in reduced affinity for target strand (TS) overhangs to the catalytic site and significantly increased trans-cleavage efficiency by up to 5.8-fold. In addition, we used a novel salt dilution method to exploit the enhanced trans-cleavage activity of Cas12a under low ionic strength conditions (7-fold), significantly improving the sensitivity of our Cas12a-based detection system. To demonstrate the clinical potential of our Cas12a-based detection system, we validated its ability to detect small amounts of hepatitis B virus (HBV) DNA model using the combination of the KE1096AA Cas12a mutant and the salt dilution method, which enables the detection of DNA at atto-molar concentrations. Our strategy to enhance the trans-cleavage activity of Cas12a paves the way for the development of more sensitive and efficient Cas12a-based diagnostics.

Sustained activation of the FGF1-MEK-ERK pathway inhibits proliferation, invasion and migration and enhances radiosensitivity in mouse angiosarcoma cells.

Angiosarcoma is a rare refractory soft-tissue tumor with a poor prognosis and is treated by radiotherapy. The fibroblast growth factor 1 (FGF1) mutant, with enhanced thermostability due to several substituted amino acids, inhibits angiosarcoma cell metastasis, yet the mechanism of action is unclear. This study aims to clarify the FGF1 mutant mechanism of action using ISOS-1 mouse angiosarcoma cells. The wild-type FGF1 or FGF1 mutant was added to ISOS-1 cells and cultured, evaluating cell numbers over time. The invasive and migratory capacity of ISOS-1 cells was assessed by transwell analysis. ISOS-1 cell radiosensitivity was assessed by colony formation assay after X-ray irradiation. To examine whether mitogen-activated protein kinase (MEK) inhibitor counteracts the FGF1 mutant effects, a combination of MEK inhibitor and FGF1 mutant was added to ISOS-1 cells and cultured. The FGF1 mutant was observed to inhibit ISOS-1 cell proliferation, invasion and migration by sustained FGF1 signaling activation. A MEK inhibitor suppressed the FGF1 mutant-induced inhibition of proliferation, invasion and migration of ISOS-1 cells. Furthermore, the FGF1 mutant enhanced radiosensitivity of ISOS-1 cells, but MEK inhibition suppressed the increased radiosensitivity. In addition, we found that the FGF1 mutant strongly inhibits actin polymerization, suggesting that actin cytoskeletal dynamics are closely related to ISOS-1 cell radiosensitivity. Overall, this study demonstrated that in ISOS-1 cells, the FGF1 mutant inhibits proliferation, invasion and migration while enhancing radiosensitivity through sustained activation of the MEK-mediated signaling pathway.

Abstract 6056: Identification of first-in-class, orally bioavailable SOS1 bifunctional degraders for the treatment of KRAS- and RTK-driven cancers

Abstract Monotherapies which target nodes within the RTK-RAS-MAPK pathway often display early potency in the clinic, but their preliminary response is limited as tumors begin to regrow due to adaptive and acquired resistance mechanisms. Disrupting proteins that mitigate either type of resistance mechanism in combination with oncogene-targeted agents could help extend and enhance the clinical potential of these drugs. Son of Sevenless 1 (SOS1), a RAS guanine nucleotide exchange factor, is a highly attractable protein to target given its role in transducing inputs from upstream receptor tyrosine kinases (RTKs) to downstream RAS proteins to regulate signaling pathways involved in cell proliferation and survival. Additionally, SOS1 is subject to MAPK-mediated negative feedback inhibition and drugs targeting upstream and downstream nodes relieves SOS1 from this inhibition resulting in pathway reactivation. To address this need, we have developed orally bioavailable SOS1 bifunctional degraders for cancers driven by KRAS and RTKs. Our top two lead molecules demonstrate CRBN- and proteasomal-mediated SOS1 degradation with DC50s < 10nM and maximum degradation > 90%. Consistent with this notion, SOS1 degradation inhibited downstream signaling marker phopshoERK with IC50s < 5nM and were 26- or 53-fold more potent when compared to the clinical SOS1 inhibitor. Notably, the SOS1 degraders exhibited weaker SOS1 binding compared to other SOS1 inhibitors highlighting that the potency is heavily driven by the catalytic nature of degradation rather than occupancy-driven events. Both SOS1 degraders displayed CRBN- and SOS1-dependent antiproliferative activity in KRAS- and EGFR- mutant, solid tumor cell lines as well as in BCR-ABL driven, CML cells with IC50 values ranging from 0.5-10nM. Consistent with our in vitro data, oral administration of our SOS1 degraders in mice harboring H358 (KRAS G12C) and H441 (KRAS G12V) xenografts resulted in SOS1 degradation, phosphoERK reduction and significant tumor growth inhibition. While SOS1 degrader monotherapy demonstrated in vitro and in vivo potency, combination of mutant or allele specific EGFR and KRAS inhibitors and ABL inhibitors with our proof-of-concept degraders prevented pathway reactivation associated with these inhibitors and lead to a more durable response. These combinations also yielded synergistic effects in in vitro proliferation assays as well as enhanced tumor growth inhibition in KRAS-mutant xenograft models. Together, our SOS1 degraders alone and in combination with other targeted agents demonstrated anti-tumor activity in a variety of KRAS- and RTK-driven cancer cell lines and xenograft models. These results support the potential of orally bioavailable SOS1 degraders to help mitigate pathway reactivation for enhanced efficacy and prolonged response duration. Citation Format: Kyle Begovich, Angela Schoolmeesters, Navin Rajapakse, Elena Martinez, Qiao Liu, Arvind Shakya, Akinori Okano, Venkat Mali, Shenlin Huang, Aparajita Chourasia, Leah Fung. Identification of first-in-class, orally bioavailable SOS1 bifunctional degraders for the treatment of KRAS- and RTK-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6056.

Polymorphism analysis of drug resistance markers in Plasmodium falciparum isolates from Benin

Like most countries in sub-Saharan African countries, Benin continues to bear a heavy malaria burden. In 2014, the National Malaria Control Programme (NMCP) changed its treatment policy, and recommended the use of artemisinin-based combination therapy (ACT) as first-line treatment for uncomplicated Plasmodium falciparum cases. The study presented here was conducted to investigate the impact of current antimalarial drug resistance on the country. Molecular surveillance targeting the Pfcrt, Pfmdr1, Pfkelch13, dhfr, and dhps genes was carried out on samples from patients positive for P. falciparum malaria by microscopy, LAMP and PCR diagnostic test. Molecular analysis was performed using targeted amplicon deep sequencing (TADS). In addition, the frequency of parasites with dual deletion of the histidine-rich protein 2 and 3 genes (pfhrp2 and pfhrp3), known to be responsible of the performance of HRP-based malaria rapid diagnostic tests (HRP-RDT), was estimated. Fifty-three falciparum samples collected at the Saint Jean de Dieu hospital in Tanguiéta, Benin, were tested. No Pfkelch13 validated or candidate artemisinin partial resistant variants were identified. A marked prevalence of Asn51Ile (N51I), Cys59Arg (C59R), and Ser108Asn (S108N) mutant alleles was found in the dhfr gene, representing the most frequent genotype (64%). Five-point mutations were detected in dhps, Ile431Val (I431V), Ser436Ala (S436A), Ala437Gly (A437G), Ala581Gly (A581G), Ala613Ser (A613S) of which the third was the most common (92%). No mutation was identified in dhps Lys540Glu (K540E). The quintuple mutant genotype resulting from the combination of the dhfr triple mutant (51I/59R/108N) with the dhps double mutant 436A/437G was detected at a frequency of 30%. Low levels of mutations in Pfcrt and no mutation at codon 86 in the Pfmdr1 DNA fragment were observed, whereas a high level of Tyr184Phe (Y184F) polymorphism in the Pfmdr1 gene was found. These results could be indicative, over a decade after the implementation of ACT therapy, of the return of chloroquine-sensitive but artemether-lumefantrine resistant falciparum genotypes in Benin. There was no evidence of HRP2 and HRP3 deletions. Data from the present study support the need for routine monitoring of molecular markers of antimalarial drug resistance as part of surveillance activities aimed to make informed treatment policy decisions at the national level.

Association of Increased Programmed Death Ligand 1 Expression and Regulatory T Cells Infiltration with Higher Hepatocellular Carcinoma Recurrence in Patients with Hepatitis B Virus Pre-S2 Mutant after Curative Surgical Resection.

Although surgical resection is available as a potentially curative therapy for hepatocellular carcinoma (HCC), high recurrence of HCC after surgery remains a serious obstacle for long-term patient survival. Therefore, the discovery of valuable prognostic biomarkers for HCC recurrence is urgently needed. Pre-S2 mutant is a mutant form of hepatitis B virus (HBV) large surface protein which is expressed from the HBV surface gene harboring deletion mutations spanning the pre-S2 gene segment. Pre-S2 mutant-positive HCC patients have been regarded as a high-risk population of HCC recurrence after resection surgery and display increased immune checkpoint programmed death ligand 1 (PD-L1) expression and pro-tumor regulatory T cells (Tregs) infiltration in tumor tissues. In this study, the association of higher levels of PD-L1 expression and Tregs infiltration in tumor tissues with post-operative HCC recurrence in pre-S2 mutant-positive HCC patients was evaluated. We found that patients with pre-S2 mutant in combination with higher levels of PD-L1 expression and Tregs infiltration in tumor tissues were independently associated with a higher risk of HCC recurrence (hazard ratio, 4.109; p value = 0.0011) and poorer recurrence-free survival (median, 8.2 versus 18.0 months; p value = 0.0004) than those of patients with either one or two of these three biomarkers. Furthermore, a combination of pre-S2 mutant, intra-tumoral PD-L1 expression, and tumor-infiltrating Tregs exhibited superior performance in identifying patients at a higher risk of HCC recurrence (area under the receiver operating characteristic curve, 0.8400). Collectively, this study suggests that higher levels of PD-L1 expression and Tregs infiltration in tumor tissues predicted a higher risk of HCC recurrence in pre-S2 mutant-positive HCC patients after curative surgical resection.

A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6'-Amino-Analogs for Bioimaging Purposes.

Beetle luciferases produce bioluminescence (BL) colors ranging from green to red, having been extensively used for many bioanalytical purposes, including bioimaging of pathogen infections and metastasis proliferation in living animal models and cell culture. For bioimaging purposes in mammalian tissues, red bioluminescence is preferred, due to the lower self-absorption of light at longer wavelengths by hemoglobin, myoglobin and melanin. Red bioluminescence is naturally produced only by Phrixothrix hirtus railroad worm luciferase (PxRE), and by some engineered beetle luciferases. However, Far-Red (FR) and Near-Infrared (NIR) bioluminescence is best suited for bioimaging in mammalian tissues due to its higher penetrability. Although some FR and NIR emitting luciferin analogs have been already developed, they usually emit much lower bioluminescence activity when compared to the original luciferin-luciferases. Using site-directed mutagenesis of PxRE luciferase in combination with 6′-modified amino-luciferin analogs, we finally selected novel FR combinations displaying BL ranging from 636–655 nm. Among them, the combination of PxRE-R215K mutant with 6′-(1-pyrrolidinyl)luciferin proved to be the best combination, displaying the highest BL activity with a catalytic efficiency ~2.5 times higher than the combination with native firefly luciferin, producing the second most FR-shifted bioluminescence (650 nm), being several orders of magnitude brighter than commercial AkaLumine with firefly luciferase. Such combination also showed higher thermostability, slower BL decay time and better penetrability across bacterial cell membranes, resulting in ~3 times higher in vivo BL activity in bacterial cells than with firefly luciferin. Overall, this is the brightest FR emitting combination ever reported, and is very promising for bioimaging purposes in mammalian tissues.

Enzyme-linked immunosorbent assays using virus-like particles containing mutations of conserved residues on envelope protein can distinguish three flavivirus infections

ABSTRACT The recent outbreaks of Zika virus (ZIKV) in flavivirus-endemic regions highlight the need for sensitive and specific serological tests. Previously we and others reported key fusion loop (FL) residues and/or BC loop (BCL) residues on dengue virus (DENV) envelope protein recognized by flavivirus cross-reactive human monoclonal antibodies and polyclonal sera. To improve ZIKV serodiagnosis, we employed wild type (WT) and FL or FL/BCL mutant virus-like particles (VLP) of ZIKV, DENV1 and West Nile virus (WNV) in enzyme linked immunosorbent assays (ELISA), and tested convalescent-phase serum or plasma samples from reverse-transcription PCR-confirmed cases with different ZIKV, DENV and WNV infections. For IgG ELISA, ZIKV WT-VLP had a sensitivity of 100% and specificity of 52.9%, which was improved to 83.3% by FL/BCL mutant VLP and 92.2% by the ratio of relative optical density of mutant to WT VLP. Similarly, DENV1 and WNV WT-VLP had a sensitivity/specificity of 100%/70.0% and 100%/56.3%, respectively; the specificity was improved to 93.3% and 83.0% by FL mutant VLP. For IgM ELISA, ZIKV, DENV1 and WNV WT-VLP had a specificity of 96.4%, 92.3% and 91.4%, respectively, for primary infection; the specificity was improved to 93.7–99.3% by FL or FL/BCL mutant VLP. An algorithm based on a combination of mutant and WT-VLP IgG ELISA is proposed to discriminate primary ZIKV, DENV and WNV infections as well as secondary DENV and ZIKV infection with previous DENV infections; this could be a powerful tool to better understand the seroprevalence and pathogenesis of ZIKV in regions where multiple flaviviruses co-circulate.

Field Performance of High Oleic Soybeans with Mutant FAD2‐1A and FAD2‐1B Genes in Tennessee

AbstractSoybean [Glycine max (L.) Merr.] oil with high oleic acid (>75%) has increased oxidative stability and health benefits that are valuable for food, fuel, and industrial products. It has been determined that two naturally occurring mutations in genes FAD2‐1A and FAD2‐1B can combine to produce high oleic soybeans. The objective of this study was to test the effect of these mutant alleles on seed yield and oil and protein concentration. Molecular markers assisted in the creation of a population of 48 BC3F2:4 lines (93.75% expected genome commonality). Each line was classified into one of four genotypic groups where both FAD2‐1A and FAD2‐1B genes were either homozygous wild type or mutant, respectively. Twelve lines for each genotypic group were evaluated in three replications at six locations across Tennessee. There was no seed yield difference between the high oleic genotypic group and the other groups (P < 0.05). On the other hand, there were differences in fatty acid profiles and oil and protein concentrations. In combination, the mutant FAD2‐1A and FAD2‐1B alleles produced a mean of 803.1 g kg−1 oleic acid. This is, on average, approximately 500 g kg−1 more oleic acid compared to soybean lines with only one mutant FAD2‐1 allele. The high oleic double mutant group had more total oil (228.0 g kg−1) and protein (401.0 g kg−1) compared to all other genotypic groups (P < 0.05). Overall, this specific combination of mutant FAD2‐1A and FAD2‐1B alleles appears to generate conventional high oleic soybeans without a yield drag.

Quantitative CT imaging features correlate with colorectal cancer liver metastases (CLM) mutational status

Background: It has been demonstrated that CLM tumor mutational status correlates with survival after surgical resection. However, preoperative prognostication is challenged by a lack of imaging features associated with adverse mutations. Utilizing patient cohorts with and without the deleterious combination of mutant KRAS/APC/TP53, this study sought quantitative biomarkers from CT image analysis. Methods: A training cohort of patients with genotyped CLM (25 patients with triple-mutant and 14 with triple-wildtype tumors) was identified. Preoperative multiphasic CT images were used to extract quantitative imaging features including image gradients, co-occurrence matrices, and pixel neighborhood statistics. Pairwise analyses were performed, and independent t-tests were used to compare the means of the identified imaging features. A random forest classifier was built to determine an integrated imaging signature for triple-mutant and wildtype patients. Results: At the time of imaging, the two cohorts shared similar distributions of demographic, clinicopathologic, and therapeutic features. This included the presence of other (non- KRAS/APC/TP53) mutations and microsatellite instability, as well as, the number and size of tumors resected at subsequent surgery. Imaging feature analyses of the pairwise differences between mutant and wildtype images were found to be statistically significant (p < 0.05), with top features including skewness (p = 0.02), energy (p = 0.03), and entropy (p = 0.03). In-sample training accuracy for prediction of tumor mutation status was 66%. Conclusion: This study is the first to demonstrate the ability of quantitative CT imaging analysis to differentiate between CLM with and without KRAS/APC/TP53 mutations. Validation of this important genotype-phenotype relationship in a larger patient cohort with mixed mutational status is ongoing.

Structural heterogeneity and intersubject variability of Aβ in familial and sporadic Alzheimer’s disease

Point mutations in the amyloid-β (Aβ) coding region produce a combination of mutant and WT Aβ isoforms that yield unique clinicopathologies in familial Alzheimer's disease (fAD) and cerebral amyloid angiopathy (fCAA) patients. Here, we report a method to investigate the structural variability of amyloid deposits found in fAD, fCAA, and sporadic AD (sAD). Using this approach, we demonstrate that mutant Aβ determines WT Aβ conformation through prion template-directed misfolding. Using principal component analysis of multiple structure-sensitive fluorescent amyloid-binding dyes, we assessed the conformational variability of Aβ deposits in fAD, fCAA, and sAD patients. Comparing many deposits from a given patient with the overall population, we found that intrapatient variability is much lower than interpatient variability for both disease types. In a given brain, we observed one or two structurally distinct forms. When two forms coexist, they segregate between the parenchyma and cerebrovasculature, particularly in fAD patients. Compared with sAD samples, deposits from fAD patients show less intersubject variability, and little overlap exists between fAD and sAD deposits. Finally, we examined whether E22G (Arctic) or E22Q (Dutch) mutants direct the misfolding of WT Aβ, leading to fAD-like plaques in vivo. Intracerebrally injecting mutant Aβ40 fibrils into transgenic mice expressing only WT Aβ induced the deposition of plaques with many biochemical hallmarks of fAD. Thus, mutant Aβ40 prions induce a conformation of WT Aβ similar to that found in fAD deposits. These findings indicate that diverse AD phenotypes likely arise from one or more initial Aβ prion conformations, which kinetically dominate the spread of prions in the brain.

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called “electrostatic gatekeeper effect”. This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the “electrostatic gatekeeper effect”. These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

Synergistic activity of Card11 mutant and Bcl6 in the development of diffuse large B-cell lymphoma in a mouse model.

Diffuse large B‐cell lymphoma (DLBCL) is the most common subtype of malignant lymphoma; it derives from germinal center B cells. Although DLBCL harbors many genetic alterations, synergistic roles between such alterations in the development of lymphoma are largely undefined. We previously established a mouse model of lymphoma by transplanting gene‐transduced germinal center B cells into mice. Here, we chose one of the frequently mutated genes in DLBCL, Card11 mutant, to explore its possible synergy with other genes, using our lymphoma model. Given that BCL6 and BCL2 expression and/or function are often deregulated in human lymphoma, we examined the possible synergy between Card11, Bcl6, and Bcl2. Germinal center B cells were induced in vitro, transduced with Card11 mutant, Bcl6, and Bcl2, and transplanted. Mice rapidly developed lymphomas, with exogenously transduced Bcl2 being dispensable. Although some mice developed lymphoma in the absence of transduced Bcl6, the absence was compensated by elevated expression of endogenous Bcl6. Additionally, the synergy between Card11 mutant and Bcl6 in the development of lymphoma was confirmed by the fact that the combination of Card11 mutant and Bcl6 caused lymphoma or death significantly earlier and with higher penetrance than Card11 mutant or Bcl6 alone. Lymphoma cells expressed interferon regulatory factor 4 and PR domain 1, indicating their differentiation toward plasmablasts, which characterize activated B cell‐like DLBCL that represents a clinically aggressive subtype in humans. Thus, our mouse model provides a versatile tool for studying the synergistic roles of altered genes underlying lymphoma development.

Combination of Null, Variant, and Mutant Wx Alleles in Common Wheat Leads to Amylose Variations Ranging from Waxy to Normal

The apparent amylose contents due to five variant or mutant Wx alleles (Wx‐A1c, ‐A1i, ‐D1f, ‐D1 g, and ‐D1h) in common wheat (Triticum aestivum L.) were analyzed. The Wx gene encodes the granule‐bound starch synthase that produces amylose in grain starch. One wheat line was used for each Wx genotype and the genetic backgrounds of the lines were not identical. The wheat lines were ranked, from lowest apparent amylose content to highest, as follows: waxy Bandowase (Wx‐A1b, ‐B1b, ‐D1b, abbreviated as bbb), Tanikei A6599‐4 (bbf), Wx‐A1i (ibb), Wx‐A1c (cbb), K107Afpp4 (bbg), Tanikei A6099 (bbh), Wx‐A1a (abb), Wx‐D1a (bba), and Bandowase (aaa, normal amylose level: approximately 30%). The blue value and λmax of the lines were largely consistent with their rank based on apparent amylose content. A line carrying two variant alleles (ibf) produced an apparent amylose content between those of Wx‐A1i (ibb) and Wx‐A1c (cbb), and a line with cbf produced an apparent amylose content between those of Wx‐A1c and Wx‐A1a (abb). Other studies have reported that five genotypes exist between abb and aaa. Although we used only one line for each genotype, the amylose contents of wheat developed using three null and five variant Wx alleles should range from waxy in bbb to normal in aaa with approximate continuity. The mutant allele Wx‐D1g carried a C‐to‐T mutation, which changed proline to leucine at the C terminus of the protein. This explains the decrease in amylose content caused by the Wx‐D1g allele.

P212A Mutant of Dihydrodaidzein Reductase Enhances (S)-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System.

(S)-Equol, a gut bacterial isoflavone derivative, has drawn great attention because of its potent use for relieving female postmenopausal symptoms and preventing prostate cancer. Previous studies have reported on the dietary isoflavone metabolism of several human gut bacteria and the involved enzymes for conversion of daidzein to (S)-equol. However, the anaerobic growth conditions required by the gut bacteria and the low productivity and yield of (S)-equol limit its efficient production using only natural gut bacteria. In this study, the low (S)-equol biosynthesis of gut microorganisms was overcome by cloning the four enzymes involved in the biosynthesis from Slackia isoflavoniconvertens into Escherichia coli BL21(DE3). The reaction conditions were optimized for (S)-equol production from the recombinant strain, and this recombinant system enabled the efficient conversion of 200 μM and 1 mM daidzein to (S)-equol under aerobic conditions, achieving yields of 95% and 85%, respectively. Since the biosynthesis of trans-tetrahydrodaidzein was found to be a rate-determining step for (S)-equol production, dihydrodaidzein reductase (DHDR) was subjected to rational site-directed mutagenesis. The introduction of the DHDR P212A mutation increased the (S)-equol productivity from 59.0 mg/liter/h to 69.8 mg/liter/h in the whole-cell reaction. The P212A mutation caused an increase in the (S)-dihydrodaidzein enantioselectivity by decreasing the overall activity of DHDR, resulting in undetectable activity for (R)-dihydrodaidzein, such that a combination of the DHDR P212A mutant with dihydrodaidzein racemase enabled the production of (3S,4R)-tetrahydrodaidzein with an enantioselectivity of >99%.

Abstract 4453: Synergistic anticancer effect of combination of AZD6244 and BYL719 targeting KRAS mutant in non-small cell lung cancer

Abstract KRAS mutation is found in 10-30% of non-small cell lung cancer (NSCLC) and highly correlated with smokers. So far, targeted agents demonstrating therapeutic activity against K-ras mutation are not available in a clinical setting. In this study, we show that dual agents, MEK inhibitor, AZD6244, and PI3K inhibitor, BYL719, have a synergistic effect on only KRAS mutant NSCLC cells. We defined that the combination of AZD6244 and BYL719 at ratios of IC50 1/2:1/2, 1:1, 2:2 (μM / μM) has ratio-dependent synergistic effect. In vitro, the combination of AZD6244 and BYL719 potently inhibited cell proliferation with cytotoxicity in H2009 and A549 cells more than the same cells treated with single drug. Also, BYL719 enhanced AZD6244-induced apoptosis and the combination treatment induced cleavage-PARP and Bim overexpression. In vivo, the antitumor efficacy was potentiated by co-treatment of AZD6244 and BYL719 in A549 xenografts. These data suggest that the combination of MEK and PI3K inhibitors might be a candidate for clinical development for its synergistic effect against KRAS-driven lung cancer. Citation Format: Eun Hye Jho, Sin Il Kang, Yeon-Hee Bae, Young Mee Park, Jong-Mu Sun, Jin Seok Ahn, Keunchil Park, Myung-Ju Ahn. Synergistic anticancer effect of combination of AZD6244 and BYL719 targeting KRAS mutant in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4453. doi:10.1158/1538-7445.AM2014-4453

Bacillus anthracisprotease InhA regulates BslA‐mediated adhesion in human endothelial cells

To achieve widespread dissemination in the host, Bacillus anthracis cells regulate their attachment to host endothelium during infection. Previous studies identified BslA (Bacillus anthracis S-layer Protein A), a virulence factor of B. anthracis, as necessary and sufficient for adhesion of vegetative cells to human endothelial cells. While some factors have been identified, bacteria-specific contributions to BslA mediated adhesion remain unclear. Using the attenuated vaccine Sterne 7702 strain of B. anthracis, we tested the hypothesis that InhA (immune inhibitor A), a B. anthracis protease, regulates BslA levels affecting the bacteria's ability to bind to endothelium. To test this, a combination of inhA mutant and complementation analysis in adhesion and invasion assays, Western blot and InhA inhibitor assays were employed. Results show InhA downregulates BslA activity reducing B. anthracis adhesion and invasion in human brain endothelial cells. BslA protein levels in ΔinhA bacteria were significantly higher than wild-type and complemented strains showing InhA levels and BslA expression are inversely related. BslA was sensitive to purified InhA degradation in a concentration- and time-dependent manner. Taken together these data support the role of InhA regulation of BslA-mediated vegetative cell adhesion and invasion.

Association of syntenin-1 with M-RIP polarizes Rac-1 activation during chemotaxis and immune interactions

In this study, we describe that the PDZ protein syntenin-1 is a crucial element for the generation of signaling asymmetry during the cellular response to polarized extracellular cues. We analyze the role of syntenin-1 in the control of asymmetry in two independent models of T cell polarization--the migratory response to chemoattractants and the establishment of cognate interactions between T cells and antigen-presenting cells (APCs). A combination of mutant, biochemical and siRNA approaches demonstrate that syntenin-1 is vital for the generation of polarized actin structures such as the leading edge and the contact zone with APCs. We found that the mechanism by which syntenin-1 controls actin polymerization relies on its mandatory role for activation of the small GTPase Rac. Syntenin-1 controls Rac through a specific association with the myosin phosphatase Rho interacting protein (M-RIP), which occurs in response to phosphorylation of syntenin-1 by Src at Tyr4. Our data indicate the key role of syntenin-1 in the generation of functional asymmetry in T cells and provide a novel mechanistic link between receptor activation and actin polymerization and accumulation in response to extracellular stimulation.

Output Targets and Transcriptional Regulation by a Cyclic Dimeric GMP-Responsive Circuit in the Vibrio parahaemolyticus Scr Network

The Vibrio parahaemolyticus Scr system modulates decisions pertinent to surface colonization by affecting the cellular level of cyclic dimeric GMP (c-di-GMP). In this work, we explore the scope and mechanism of this regulation. Transcriptome comparison of ΔscrABC and wild-type strains revealed expression differences with respect to ∼100 genes. Elevated c-di-GMP repressed genes in the surface-sensing regulon, including those encoding the lateral flagellar and type III secretion systems and N-acetylglucosamine-binding protein GpbA while inducing genes encoding other cell surface molecules and capsular polysaccharide. The transcription of a few regulatory genes was also affected, and the role of one was characterized. Mutations in cpsQ suppressed the sticky phenotype of scr mutants. cpsQ encodes one of four V. parahaemolyticus homologs in the CsgD/VpsT family, members of which have been implicated in c-di-GMP signaling. Here, we demonstrate that CpsQ is a c-di-GMP-binding protein. By using a combination of mutant and reporter analyses, CpsQ was found to be the direct, positive regulator of cpsA transcription. This c-di-GMP-responsive regulatory circuit could be reconstituted in Escherichia coli, where a low level of this nucleotide diminished the stability of CpsQ. The molecular interplay of additional known cps regulators was defined by establishing that CpsS, another CsgD family member, repressed cpsR, and the transcription factor CpsR activated cpsQ. Thus, we are developing a connectivity map of the Scr decision-making network with respect to its wiring and output strategies for colonizing surfaces and interaction with hosts; in doing so, we have isolated and reproduced a c-di-GMP-sensitive regulatory module in the circuit.

Chronic Intestinal Inflammation Induces Stress-Response Genes in Commensal Escherichia coli

Intestinal microbes induce homeostatic mucosal immune responses, but can also cause inappropriate immune activation in genetically susceptible hosts. Although immune responses to bacterial products have been studied extensively, little is known about how intestinal inflammation affects functions of commensal luminal microbes. Microarrays and real-time polymerase chain reaction were used to profile transcriptional changes in luminal bacteria from wild-type and IL-10(-/-) mice monoassociated with a nonpathogenic, murine isolate of Escherichia coli (NC101, which causes colitis in gnotobiotic IL-10(-/-) mice). Colonic inflammation and innate and adaptive immune responses were measured in wild-type and IL-10(-/-) mice monoassociated with mutant NC101 that lack selected, up-regulated genes, and in IL-10(-/-) mice that were colonized with a combination of mutant and parental NC101. We measured intracellular survival of bacteria within primary macrophages from mice and resulting production of tumor necrosis factor. Bacteria from IL-10(-/-) mice with colitis had significant up-regulation of the stress-response regulon, including the small heat shock proteins IbpA and IbpB that protect E coli from oxidative stress, compared to healthy, wild-type controls. In IL-10(-/-) mice, expression of ibpAB reduced histologic signs of colon inflammation, secretion of interleukin-12/23p40 in colonic explant cultures, serologic reactivity to NC101 antigens, and secretion of interferon-gamma by stimulated mesenteric lymph node cells. Infection of primary macrophages by bacteria that express ibpAB was associated with decreased intracellular survival and reduced secretion of tumor necrosis factor. Chronic intestinal inflammation causes functional alterations in gene expression in commensal gut bacterium (E coli NC101). Further studies of these expression patterns might identify therapeutic targets for patients with inflammatory bowel diseases.

Functional analysis of Streptococcus pyogenes nuclease A (SpnA), a novel group A streptococcal virulence factor

Streptococcus pyogenes nuclease A (SpnA) is a recently discovered DNase that plays a role in virulence as shown in a mouse infection model. SpnA is the only cell wall-anchored DNase found in S. pyogenes thus far and shows a unique protein architecture. The C-terminal nuclease domain contains highly conserved catalytic site and Mg(2+) binding site residues. However, expression of the SpnA nuclease domain alone resulted in a soluble, but enzymatically inactive protein. We found that at least two out of three oligonucleotide/oligosaccharide-binding fold motifs found in the N-terminal domain are required for SpnA activity, probably contributing to substrate binding. Using a combination of a spnA deletion mutant and a Lactococcus lactis'gain-of-function' mutant, we have shown that SpnA promotes survival in whole human blood and in neutrophil killing assays and this is, at least in part, achieved by the destruction of neutrophil extracellular traps (NETs). We observed higher frequencies for anti-SpnA antibodies in streptococcal disease patient sera (79%, n = 19) compared with sera from healthy donors (33%, n = 9) suggesting that SpnA is expressed during infection. Detection of anti-SpnA antibodies in patient serum might be useful for the diagnostic of post-streptococcal diseases, such as acute rheumatic fever or glomerulonephritis.