High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library

In antibody discovery, in-depth analysis of an antibody library and high-throughput retrieval of clones in the library are crucial to identifying and exploiting rare clones with different properties. However, existing methods have technical limitations, such as low process throughput from the laborious cloning process and waste of the phenotypic screening capacity from unnecessary repetitive tests on the dominant clones. To overcome the limitations, we developed a new high-throughput platform for the identification and retrieval of clones in the library, TrueRepertoire?. This new platform provides highly accurate sequences of the clones with linkage information between heavy and light chains of the antibody fragment. Additionally, the physical DNA of clones can be retrieved in high throughput based on the sequence information. We validated the high accuracy of the sequences and demonstrated that there is no platform-specific bias. Moreover, the applicability of TrueRepertoire? was demonstrated by a phage-displayed single-chain variable fragment library targeting human hepatocyte growth factor protein.     

Computational approach to site-directed ligand discovery

A computational approach, Systematic Conformational Search & Induced Fit (SCI&FI), to site-directed ligand discovery (Tethering) is presented. SCI&FI has the ability to predict the binding site, binding mode, and bound dynamics of small molecule fragments covalently tethered to a protein. The SCI&FI method was engineered with the ability to model induced fit conformational changes of the protein because of the binding of the tether. SCI&FI generates comprehensive picture of the binding preferences of the tether to the protein by elucidating potential binding sites of the tether and by describing regions of receptor space capable of conformational change because of the binding of the tether. The SCI&FI method provides a complementary approach to experimental tethering. Initial validation of the SCI&FI method is reported by predicting the 3D structure of two Interleukin-2 and an Interleukin-4 tethered-protein systems.     

Molecular design,synthesis and in vitro biological evaluation of thienopyrimidine–hydroxamic acids as chimeric kinase HDAC inhibitors: a challenging approach to combat cancer

A series of thieno[2,3-d]pyrimidine-based hydroxamic acid hybrids was designed and synthesised as multitarget anti-cancer agents, through incorporating the pharmacophore of EGFR, VEGFR2 into the inhibitory functionality of HDAC6. Three compounds (12c, 15b and 20b) were promising hits, whereas (12c) exhibited potent VEGFR2 inhibition (IC₅₀=185 nM), potent EGFR inhibition (IC₅₀=1.14 µM), and mild HDAC6 inhibition (23% inhibition). Moreover, compound (15c) was the most potent dual inhibitor among all the synthesised compounds, as it exhibited potent EGFR and VEGFR2 inhibition (IC₅₀=19 nM) and (IC₅₀=5.58 µM), respectively. While compounds (20d) and (7c) displayed nanomolar selective kinase inhibition with EGFR IC₅₀= 68 nM and VEGFR2 IC₅₀= 191 nM, respectively. All of the synthesised compounds were screened in vitro for their cytotoxic effect on 60 human NCI tumour cell lines. Additionally, molecular docking studies and ADMET studies were carried out to gain further insight into their binding mode and predict the pharmacokinetic properties of all the synthesised inhibitors.     

Small Molecule Sequestration of the Intrinsically Disordered Protein,p27Kip1, Within Soluble Oligomers

Proteins that exhibit intrinsically disordered regions (IDRs) are prevalent in the human proteome and perform diverse biological functions, including signaling and regulation. Due to these important roles, misregulation of intrinsically disordered proteins (IDPs) is associated with myriad human diseases, including neurodegeneration and cancer. The inherent flexibility of IDPs limits the applicability of the traditional structure-based drug design paradigm; therefore, IDPs have long been considered “undruggable”. Using NMR spectroscopy and other methods, we previously discovered small, drug-like molecules that bind specifically, albeit weakly, to dynamic clusters of aromatic residues within p27^Kip1 (p27), an archetypal disordered protein involved in cell cycle regulation. Here, using synthetic chemistry, NMR spectroscopy and other biophysical methods, we discovered elaborated analogs of our previously reported molecules with 30-fold increased affinity for p27 (apparent K_d = 57 ± 19 μM). Strikingly, using analytical ultracentrifugation methods, we showed that the highest affinity compounds caused p27 to form soluble, disordered oligomers. Based on these observations, we propose that sequestration within soluble oligomers may represent a general strategy for therapeutically targeting disease-associated IDPs in the future.     

Investigation of Vetiver Grass Capability in Phytoremediation of Contaminated Soils with Heavy Metals (Pb,Cd, Mn,and Ni)

ABSTRACT

Soil contamination with heavy metals has become a worldwide concern. A sustainable technology to mitigate heavy metal contamination is extremely important. Phytoremediation is a cost-effective method, environmentally friendly, and esthetically pleasing. The aim of this study was to investigate the potential of Vetiver phytoremediation of soils contaminated with heavy metals. This research was conducted as a factorial design with four different heavy metals (lead, cadmium, manganese, and nickel) with three varying levels and also three replications for each treatment. Statistical analysis of data was performed using SPSS19 software and analysis of variance, Duncan and Pearson correlation tests. The results showed that, the highest uptake rate was related to lead metal with 282.45 mg/kg of dry soil and 83.4% uptake percentage. Then, the mean and percentage of adsorption for cadmium, nickel and manganese were 248.3 mg/kg (53.2%), 69.4 mg/kg (65.5%), and 63.29 mg/kg (61%), respectively. Lead was found to be the main component of uptake by Vetiver plant. It was found that the roots of the plant have absorbed more heavy metals than the shoots. And at the roots in total 1089.05 and on average 363.01 mg/kg and at the shoots 901.19 and on average 300.39 mg/kg, the metals used were adsorbed on three levels and four treatments. The results of analysis of variance, Duncan test and Pearson correlation showed that the effect of applied treatments on lead uptake in roots and shoots increased significantly (P ≤ 0.05) with increasing levels of treatments. The biological concentration factor was more than one, and the transfer factor was close to one. Therefore, it can be used as a phytostablization plant. The results showed that Vetiver can be considered as a refining plant due to its vegetative characteristics, cost-effectiveness and high adaptation to environmental conditions.     

A cytosolic glutathione s-transferase,GST-theta from freshwater prawn Macrobrachium rosenbergii: molecular and biochemical properties

Glutathione S-transferases play an important role in cellular detoxification and may have evolved to protect cells against reactive oxygen metabolites. In this study, we report the molecular characterization of glutathione s-transferase-theta (GST-θ) from freshwater prawn Macrobrachium rosenbergii. A full length cDNA of GSTT (1417 base pairs) was isolated and characterized bioinformatically. Exposure to virus (white spot syndrome baculovirus or M. rosenbergii nodovirus), bacteria (Aeromonas hydrophila or Vibrio harveyi) or heavy metals (cadmium or lead) significantly increased the expression of GSTT (P < 0.05) in hepatopancreas. Recombinant GST-θ with monochlorobimane substrate had an optimum activity at pH 7.5 and 35 °C. Furthermore recombinant GST-θ activity was abolished by the denaturants triton X-100, Gua-HCl, Gua-thiocyanate, SDS and urea in a dose-dependent manner. Overall, the results suggest a potential role for M. rosenbergii GST-θ in detoxification and possibly conferring immune protection.     

Prediction of Soil Lead Recontamination Trends with Decreasing Atmospheric Deposition

The mathematical model of soil mixing after atmospheric surface deposition developed in Drivas et al. (2011) is expanded here and applied to a case study of soil recontamination in areas near a lead smelter in Herculaneum, Missouri. Soil lead samples collected from the yards of several residences in Herculaneum between 2001 and 2009 show that recontamination of previously remediated yards has taken place. The model is used to predict a relative soil lead recontamination trend with time, based on the remediation date and decreasing smelter emissions over time. An average scaling factor between relative and absolute soil lead levels is derived based on over 1600 data points from 24 properties, using modeled air lead levels and the remediation date for each property. The scaling factor was used to predict soil lead recontamination trends at an additional six properties that were remediated in the mid-1990s. The predicted soil lead concentration vs. time curves match the time-trends in the soil data, explaining the observations that soil lead levels increased during the 2000s for properties remediated in 2001–2002, but decreased during the same time frame for properties remediated in the 1990s. The model can be used to predict expected recontamination trends under differing air deposition scenarios and to extrapolate expected recontamination trends into the future.     

3种土壤类型下红椿幼苗对铅胁迫的光合响应

采用盆栽试验,研究钙质紫色土、酸性紫色土和冲积土3种土壤类型上生长的红椿幼苗在不同浓度(0、200、450、2 000mg·kg-1)铅(Pb)胁迫下的光合生理响应,探讨红椿在不同土壤类型下Pb污染的生态适应性,为木本植物的重金属修复提供理论依据。结果表明:(1)3种土壤类型下,红椿幼苗叶片叶绿素a、叶绿素b及叶绿素总量随Pb胁迫浓度的增大呈总体降低的趋势,叶绿素含量在钙质紫色土上受Pb胁迫的影响不大,而在酸性紫色土和冲积土上降低明显。(2)不同土壤类型的红椿光合响应曲线变化趋势十分相似,红椿幼苗叶片的净光合速率均随Pb胁迫程度的加大呈先增加后降低的趋势且差异显著,并在Pb浓度为200和2 000mg·kg-1时光合速率分别达到最大值和最小值。(3)随着Pb胁迫浓度的增大,红椿的最大光合速率、表观量子利用效率、光补偿点等光合生理指标都发生了显著改变,说明Pb胁迫严重影响红椿幼苗的光合作用。研究表明,高浓度的铅抑制红椿幼苗叶片叶绿素的合成,从而抑制光合作用的强度,且铅胁迫的浓度越高,叶绿素含量越低,光合作用受到的抑制越强;较低浓度的Pb处理(200和450mg·kg-1)对酸性紫色土和冲积土上红椿的净光合速率影响很小,而对钙质紫色土上红椿的最大净光合速率的抑制最为明显。     

Genome-wide expression analysis of rice ABC transporter family across spatio-temporal samples and in response to abiotic stresses

Although the super family of ATP-binding cassette (ABC) proteins plays key roles in the physiology and development of plants, the functions of members of this interesting family mostly remain to be clarified, especially in crop plants. Thus, systematic analysis of this family in rice (Oryza sativa), a major model crop plant, will be helpful in the design of effective strategies for functional analysis. Phylogenomic analysis that integrates anatomy and stress meta-profiling data based on a large collection of rice Affymetrix array data into the phylogenic context provides useful clues into the functions for each of the ABC transporter family members in rice. Using anatomy data, we identified 17 root-preferred and 16-shoot preferred genes at the vegetative stage, and 3 pollen, 2 embryo, 2 ovary, 2 endosperm, and 1 anther-preferred gene at the reproductive stage. The stress data revealed significant up-regulation or down-regulation of 47 genes under heavy metal treatment, 16 genes under nutrient deficient conditions, and 51 genes under abiotic stress conditions. Of these, we confirmed the differential expression patterns of 14 genes in root samples exposed to drought stress using quantitative real-time PCR. Network analysis using RiceNet suggests a functional gene network involving nine rice ABC transporters that are differentially regulated by drought stress in root, further enhancing the prediction of biological function. Our analysis provides a molecular basis for the study of diverse biological phenomena mediated by the ABC family in rice and will contribute to the enhancement of crop yield and stress tolerance.     

Evaluation of Potential Exposure to Metals in Laundered Shop Towels

We reported in 2003 that exposure to metals on laundered shop towels (LSTs) could exceed toxicity criteria. New data from LSTs used by workers in North America document the continued presence of metals in freshly laundered towels. We assessed potential exposure to metals based on concentrations of metals on the LSTs, estimates of LST usage by employees, and the transfer of metals from LST-to-hand, hand-to-mouth, and LST-to-lip, under average- or high-exposure scenarios. Exposure estimates were compared to toxicity criteria. Under an average-exposure scenario (excluding metals’ data outliers), exceedances of the California Environmental Protection Agency, U.S. Environmental Protection Agency, and the Agency for Toxic Substances and Disease Registry toxicity criteria may occur for aluminum, cadmium, cobalt, copper, iron, and lead. Calculated intakes for these metals were up to more than 400-fold higher (lead) than their respective toxicity criterion. For the high-exposure scenario, additional exceedances may occur, and high-exposure intakes were up to 1,170-fold higher (lead) than their respective toxicity criterion. A sensitivity analysis indicated that alternate plausible assumptions could increase or decrease the magnitude of exceedances, but were unlikely to eliminate certain exceedances, particularly for lead.