Dual Targeting of EGFR with PLK1 Exerts Therapeutic Synergism in Taxane-Resistant Lung Adenocarcinoma by Suppressing ABC Transporters

To overcome the limitations of chemoresistance, combination therapies using druggable targets have been investigated. Our previous studies led us to hypothesize that the downregulation of PLK1 expression or activity can be one strategy to overcome the hurdles of taxane resistance by the downregulation of ABC transporters. To explore this, various versions of PLK1 including a constitutively active version, kinase-dead form, and polo-box domain mutant were expressed in paclitaxel-resistant lung adenocarcinoma (LUADTXR). Targeting PLK1 using shRNA or non-functional mutants downregulated ABCB1, ABCC9, and ABCG2 in LUADTXR cells, which was similar to the downregulation effects from treatment with PLK1 inhibitors. The high expression of EGFR in LUAD led us to administer gefitinib, showing a markedly reduced EGFR level in LUADTXR cells. When gefitinib and PLK1 inhibitors were combined, LUADTXR cells tended to undergo apoptosis more effectively than parental cells, showing a synergistic effect on the downregulation of ABC transporters through c-Myc and AP-1. Clinical data provide evidence for the relevance between survival rates and expressions of PLK1 and EGFR in LUAD patients. Based on these results, we suggest that a combination of gefitinib and PLK1 inhibitors exerts strong synergism in LUADTXR, which helps to overcome the limitations associated with taxanes.

Selection of functional EPHB2 genotypes from ENU mutated grass carp treated with GCRV

Background N-ethyl-N-nitrosourea (ENU) mutagenesis is a useful method for the genetic engineering of plants, and the production of functional mutants in animal models including mice and zebrafish. Grass carp reovirus (GCRV) is a haemorrhagic disease of grass carp which has caused noteworthy losses in fingerlings over the last few years. To overcome this problem, we used ENU mutant grass carp in an attempt to identify functional resistance genes for future hereditary rearing projects in grass carp. Results This study used ENU-mutated grass carp to identify genetic markers associated with resistance to the haemorrhagic disease caused by GCRV. Bulked segregant analysis (BSA) was performed on two homozygous gynogenetic ENU grass carp groups who were susceptible or resistant to GCRV. This analysis identified 466,162 SNPs and 197,644 InDels within the genomes of these mixed pools with a total of 170 genes annotated in the associated region, including 49 genes with non-synonymous mutations at SNP sites and 25 genes with frame shift mutations at InDel sites. Of these 170 mutated genes, 5 randomly selected immune-related genes were shown to be more strongly expressed in the resistant group as compared to the susceptible animals. In addition, we found that one immune-related gene, EPHB2, presented with two heterozygous SNP mutations which altered the animal’s responded to GCRV disease. These SNPs were found in the intron region of EPHB2 at positions 5859 (5859G > A) and 5968 (5968G > A) and were significantly (p = 0.002, 0.003) associated with resistance to GCRV. These SNP sites were also shown to correlate with the GCRV-resistant phenotype in these ENU grass carp. We also evaluated the mortality of the different ENU fish genotypes in response to GCRV and the SNPs in EPHB2. The outcomes of these evaluations will be useful in future selections of GCRV-resistant genes for genetic breeding in grass carp. Conclusion Our results provide a proof of concept for the application of BSA-sequence analysis in detecting genes responsible for specific functional genotypes and may help to develop better methods for marker-assisted selection, especially for disease resistance in response to GCRV.

Radiotracers for examining biological functions of plants and microbes

Tracers are used for qualitative and quantitative investigation of a system. Radiotracers have a radionuclide to observe chemical or biological processes by detection of the radionuclide's decay energy. They are non-disruptive and non-destructive to living systems and can be quantified, imaged, and measured in real time, adding value. This work focuses on radiochemistry and radiotracer techniques to understand maize uptake and localization of micronutrients and the impact of Azospirillum brasilense microbial interactions on these processes. Further, it explored how such interactions can influence stress responses in maize. Finally, it examined how the natural biological functions of A. brasilense bacteria respond to light stimulus conducted through the plant tissues. In this dissertation, the efficacy of using 4-fluorophenylboronic acid (FPBA) as a boton (B) imaging agent, which is a derivative of the B deficiency mimic phenylboronic acid (PBA), was explored. It is shown that radioactively labelled [18F]FPBA (t [subscript 1/2] [equals] 110 m) accumulates at the root tip, the root elongation zone and at lateral root initiation sites in maize roots, and also translocates to the shoot where it accumulates along leaf edges. This is the first time a radiotracer has been utilized to image B in plant systems. Nutritional iron (Fe) content was explored in Azospirillum brasilense associated maize. 59Fe (t [subscript 1/2] [equals] 44.5 d) was used to trace iron uptake kinetics and allocation to leaf. In the presence of functional mutants of this bacteria, iron uptake and allocation to leaf was enhanced in maize seedlings. Maize were grown to maturity and plants associated with the bacteria had greater crop yield (kernels cob-1) and enhanced iron and protein ferritin- the bioavailable form of iron to humans- seed content. Similar studies were completed using zinc (65Zn, t¬Ω= 244 d), where it was noted that the presence of the low-auxin producing and nitrogen-fixing bacteria strain, ipdC, enhanced zinc uptake but had no enhancement effect on allocation or zinc seed filling. Carbon metabolism in response to stresses and microbial interaction was also investigated in maize with [11C]CO2 (t [subscript 1/2] [equals] 20.4 m) radiotracer. In association with A. brasilense, maize fixed more carbon dioxide, allocated more 11C-photosynthates to the roots, and produced more 11C-exudates than control maize. Metabolic differences were studied via radio-HPLC and radio-TLC to reveal association enhanced 11C flow into hydrophobic structural components and amino acids. When nitrogen stressed, non-inoculated maize exhibited a decrease in carbon dioxide fixation, root allocation of 11C-photosynthates, and decreased 11Cexudation compared to control maize. They also saw increased 11C flow into hydrophobic structural components and sugars. When inoculated with A. brasilense and subjected to nitrogen stress, the same enhancements occurred- but fixation, allocation, and exudation recovered to near control maize levels, suggesting these bacteria ameliorate some abiotic stresses. Finally, 59Fe and [11C]CO2 radiotracers were applied to the functional mutants of A. brasilense to uncover how various biological functions were impacted by light exposure. First, light transmittance from shoot to root tissues, called light piping, in maize was shown using a DSLR camera and image intensifier. Studies showed the functional mutants with biological nitrogen fixation (BNF) capacity had enhanced assimilation of 59Fe when exposed to light relative to dark treatments and greater activity of the nitrogenase enzyme as measured by acetylene reduction assay in light, with a greater response noted for red than blue light wavelengths. Carbon assimilation as [11C]CO2 and subsequent metabolism in these bacteria were also impacted by light stimulus.

Functional mutants of Azospirillum brasilense elicit beneficial physiological and metabolic responses in Zea mays contributing to increased host iron assimilation

Iron (Fe), an essential element for plant growth, is abundant in soil but with low bioavailability. Thus, plants developed specialized mechanisms to sequester the element. Beneficial microbes have recently become a favored method to promote plant growth through increased uptake of essential micronutrients, like Fe, yet little is known of their mechanisms of action. Functional mutants of the epiphytic bacterium Azospirillum brasilense, a prolific grass-root colonizer, were used to examine mechanisms for promoting iron uptake in Zea mays. Mutants included HM053, FP10, and ipdC, which have varying capacities for biological nitrogen fixation and production of the plant hormone auxin. Using radioactive iron-59 tracing and inductively coupled plasma mass spectrometry, we documented significant differences in host uptake of Fe2+/3+ correlating with mutant biological function. Radioactive carbon-11, administered to plants as 11CO2, provided insights into shifts in host usage of ‘new’ carbon resources in the presence of these beneficial microbes. Of the mutants examined, HM053 exhibited the greatest influence on host Fe uptake with increased plant allocation of 11C-resources to roots where they were transformed and exuded as 11C-acidic substrates to aid in Fe-chelation, and increased C-11 partitioning into citric acid, nicotianamine and histidine to aid in the in situ translocation of Fe once assimilated.

Effect of Asparagine Substitutions in the YXN Loop of a Class C β-Lactamase of Acinetobacter baumannii on Substrate and Inhibitor Kinetics

ABSTRACTClass C cephalosporinases are a growing threat, and inhibitors of these enzymes are currently unavailable. Studies exploring the YXN loop asparagine in theEscherichia coliAmpC, P99, and CMY-2 enzymes have suggested that interactions between C6′ or C7′ substituents on penicillins or cephalosporins and this Asn are important in determining substrate specificity and enzymatic stability. We sought to characterize the YXN loop asparagine in the clinically important ADC-7 class C β-lactamase ofAcinetobacter baumannii. Mutagenesis at the N148 position in ADC-7 yields functional mutants (N152G, -S, -T, -Q, -A, and -C) that retain cephalosporinase activity. Using standard assays, we show that N148G, -S, and -T variants possess good catalytic activity toward cefoxitin and ceftaroline but that cefepime is a poor substrate. Because N152 variants of CMY-2, another class C β-lactamase, are more readily inhibited by tazobactam due to higher rates of inactivation, we also tested if the N148 substitutions in ADC-7 would affect inactivation by sulfone inhibitors, sulbactam and tazobactam, class A β-lactamase, andA. baumanniipenicillin-binding protein (PBP) inhibitors within vitroactivity against ADC-7. The 50% inhibitory concentrations (IC50s) for tazobactam and sulbactam were improved, with 7-fold and 2-fold reductions, respectively, for the N148S variant. A homology model of the N148S ADC-7 enzyme in a Michaelis-Menten complex with tazobactam showed a loss of interaction between N148 and the sulfone moiety of the inhibitor. We postulate that this may result in more-rapid secondary ring opening of the inhibitor, as the unbound sulfone is an excellent leaving group, leading to more-rapid formation of the stable linearized inhibitor.

N152G, -S, and -T Substitutions in CMY-2 β-Lactamase Increase Catalytic Efficiency for Cefoxitin and Inactivation Rates for Tazobactam

ABSTRACTClass C cephalosporinases are a growing threat, and clinical inhibitors of these enzymes are currently unavailable. Previous studies have explored the role of Asn152 in theEscherichia coliAmpC and P99 enzymes and have suggested that interactions between C-6′ or C-7′ substituents on penicillins or cephalosporins and Asn152 are important in determining substrate specificity and enzymatic stability. We sought to characterize the role of Asn152 in the clinically important CMY-2 cephalosporinase with substrates and inhibitors. Mutagenesis of CMY-2 at position 152 yields functional mutants (N152G, -S, and -T) that exhibit improved penicillinase activity and retain cephamycinase activity. We also tested whether the position 152 substitutions would affect the inactivation kinetics of tazobactam, a class A β-lactamase inhibitor within vitroactivity against CMY-2. Using standard assays, we showed that the N152G, -S, and -T variants possessed increased catalytic activity against cefoxitin compared to the wild type. The 50% inhibitory concentration (IC50) for tazobactam improved dramatically, with an 18-fold reduction for the N152S mutant due to higher rates of enzyme inactivation. Modeling studies have shown active-site expansion due to interactions between Y150 and S152 in the apoenzyme and the Michaelis-Menten complex with tazobactam. Substitutions at N152 might become clinically important as new class C β-lactamase inhibitors are developed.