Zhong Yang

Highly Efficient Production of Soluble Proteins from Insoluble Inclusion Bodies by a Two-Step-Denaturing and Refolding Method

The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventional procedure with one denaturation step and one refolding step using three proteins with different folding properties. The refolded proteins were found to be active using in vitro tests and a bioassay. We then tested the general applicability of this method by analyzing 88 proteins from human and other organisms, all of which were expressed as inclusion bodies. We found that about 76% of these proteins were refolded with an average of >75% yield of soluble proteins. This “two-step-denaturing and refolding” (2DR) method is simple, highly efficient and generally applicable; it can be utilized to obtain active recombinant proteins for both basic research and industrial purposes.

CTCF Mediates the Cell-Type Specific Spatial Organization of the Kcnq5 Locus and the Local Gene Regulation

Chromatin loops play important roles in the dynamic spatial organization of genes in the nucleus. Growing evidence has revealed that the multivalent functional zinc finger protein CCCTC-binding factor (CTCF) is a master regulator of genome spatial organization, and mediates the ubiquitous chromatin loops within the genome. Using circular chromosome conformation capture (4C) methodology, we discovered that CTCF may be a master organizer in mediating the spatial organization of the kcnq5 gene locus. We characterized the cell-type specific spatial organization of the kcnq5 gene locus mediated by CTCF in detail using chromosome conformation capture (3C) and 3C-derived techniques. Cohesion also participated in mediating the organization of this locus. RNAi-mediated knockdown of CTCF sharply diminished the interaction frequencies between the chromatin loops of the kcnq5 gene locus and down-regulated local gene expression. Functional analysis showed that the interacting chromatin loops of the kcnq5 gene locus can repress the gene expression in a luciferase reporter assay. These interacting chromatin fragments were a series of repressing elements whose contacts were mediated by CTCF. Therefore, these findings suggested that the dynamical spatial organization of the kcnq5 locus regulates local gene expression.

Aldose reductase from Schistosoma japonicum : crystallization and structure-based inhibitor screening for discovering antischistosomal lead compounds

BackgroundSchistosomiasis is a neglected tropical disease with high morbidity and mortality in the world. Currently, the treatment of this disease depends almost exclusively on praziquantel (PZQ); however, the emergence of drug resistance to PZQ in schistosomes makes the development of novel drugs an urgent task. Aldose reductase (AR), an important component that may be involved in the schistosome antioxidant defense system, is predicted as a potential drug target.MethodsThe tertiary structure of Schistosoma japonicum AR (Sj AR) was obtained through X-ray diffraction method and then its potential inhibitors were identified from the Maybridge HitFinder library by virtual screening based on this structural model. The effects of these identified compounds on cultured adult worms were evaluated by observing mobility, morphological changes and mortality. To verify that Sj AR was indeed the target of these identified compounds, their effects on recombinant Sj AR (rSj AR) enzymatic activity were assessed. The cytotoxicity analysis was performed with three types of human cell lines using a Cell Counting Kit-8.ResultsWe firstly resolved the Sj AR structure and identified 10 potential inhibitors based on this structural model. Further in vitro experiments showed that one of the compounds, renamed as AR9, exhibited significant inhibition in the activity of cultured worms as well as inhibition of enzymatic activity of rSj AR protein. Cytotoxicity analysis revealed that AR9 had relatively low toxicity towards host cells.ConclusionsThe work presented here bridges the gap between virtual screening and experimental validation, providing an effective and economical strategy for the development of new anti-parasitic drugs. Additionally, this study also found that AR9 may become a new potential lead compound for developing novel antischistosomal drugs against parasite AR.

Genetic variation between Schistosoma japonicum lineages from lake and mountainous regions in China revealed by resequencing whole genomes.

Schistosoma infection is a major cause of morbidity and mortality worldwide. Schistosomiasis japonica is endemic in mainland China along the Yangtze River, typically distributed in two geographical categories of lake and mountainous regions. Study on schistosome genetic diversity is of interest in respect of understanding parasite biology and transmission, and formulating control strategy. Certain genetic variations may be associated with adaptations to different ecological habitats. The aim of this study is to gain insight into Schistosoma japonicum genetic variation, evolutionary origin and associated causes of different geographic lineages through examining homozygous Single Nucleotide Polymorphisms (SNPs) based on resequenced genome data. We collected S. japonicum samples from four sites, three in the lake regions (LR) of mid-east (Guichi and Tonglin in Anhui province, Laogang in Hunan province) and one in mountainous region (MR) (Xichang in Sichuan province) of south-west of China, resequenced their genomes using Next Generation Sequencing (NGS) technology, and made use of the available database of S. japonicum draft genomic sequence as a reference in genome mapping. A total of 14,575 SNPs from 2059 genes were identified in the four lineages. Phylogenetic analysis confirmed significant genetic variation exhibited between the different geographical lineages, and further revealed that the MR Xichang lineage is phylogenetically closer to LR Guich lineage than to other two LR lineages, and the MR lineage might be evolved from LR lineages. More than two thirds of detected SNPs were nonsynonymous; functional annotation of the SNP-containing genes showed that they are involved mainly in biological processes such as signaling and response to stimuli. Notably, unique nonsynonymous SNP variations were detected in 66 genes of MR lineage, inferring possible genetic adaption to mountainous ecological condition.

Genetic structure of Daphnia galeata populations in Eastern China.

This study presents the first examination of the genetic structure of Daphnia longispina complex populations in Eastern China. Only one species, D. galeata, was present across the eight investigated lakes; as identified by taxon assignment using allelic variation at 15 microsatellite loci. Three genetically differentiated D. galeata subgroups emerged independent of the type of statistical analysis applied. Thus, Bayesian clustering, discriminant analysis based on results from factorial correspondence analysis, and UPGMA clustering consistently showed that populations from two neighbouring lakes were genetically separated from a mixture of genotypes found in other lakes, which formed another two subgroups. Clonal diversity was high in all D. galeata populations, and most samples showed no deviation from Hardy-Weinberg equilibrium, indicating that clonal selection had little effect on the genetic diversity. Overall, populations did not cluster by geographical origin. Further studies will show if the observed pattern can be explained by natural colonization processes or by recent anthropogenic impact on predominantly artificial lakes.

SU-E-T-59: A Novel Multi-Beam Dynamic IMRT with Fixed-Jaw Technique for Left Breast Cancer Patients with Regional Lymph Nodes Radiotherapy

Purpose: This study was to investigate the dosimetric benefit of a novel intensity modulated radiation therapy (IMRT) technique for irradiating the left breast and regional lymph node (RLN). Methods: The breast and RLN (internal mammary node and periclavicular node) and normal tissue were contoured for 16 consecutive left-sided breast cancer patients previously treated with RT after lumpectomy. Nine equi-spaced fields IMRT (9 -field IMRT), tangential multi-beam IMRT (tangential-IMRT) and IMRT with fixed-jaw technique (FJT-IMRT) were developed and compared with three-dimensional conformal RT (3DCRT). Prescribed dose was 50 Gy in 25 fractions. Dose distributions and dose volume histograms were used to evaluate plans. Results: All IMRTs achieved similar target coverage and substantially reduced heart V30 and V20 compared to the 3DCRT. The average heart mean dose had different changes, which were 9.0Gy for 9-field IMRT, 5.7Gy for tangential-IMRT and 4.2Gy for FJT-IMRT. For the contralateral lung and breast, the 9-field IMRT has the highest mean dose; and the FJT-IMRT and tangential-IMRT had similar lower value. For the thyroid, both 9-field IMRT and FJT-IMRT had similar V30 (20% and 22%) and were significantly lower than that of 3DCRT (34%) and tangential-IMRT (46%). Moreover, the thyroid mean dose of FJT-IMRT is the lowest. For cervical esophagus and humeral head, the FJT-IMRT also had the best sparing. Conclusion: All 9-field IMRT, tangential-IMRT and FJT-IMRT had superiority for targets coverage and substantially reduced the heart volume of high dose irradiation. The FJT-IMRT showed advantages of avoiding the contralateral breast and lung irradiation and decreasing the thyroid, humeral head and cervical esophagus radiation dose at the expense of a slight monitor units (MUs) increasing.

Integrated refolding techniques for Schistosoma japonicum MTH1 overexpressed as inclusion bodies in Escherichia coli

The full-length cDNA of MTH1in Schistosoma japonicum was previously isolated. However, insoluble protein expression in Escherichia coli is the biggest bottleneck limiting biological and biophysical studies. Protein aggregation could not be significantly prevented using solubilization or refolding techniques, and denatured MTH1 protein could not be refolded to the native monomer form. Hence, integrating several refolding techniques within the protein refolding process of MTH1, a large amount of active MTH1 was obtained for protein crystallization. We primarily utilized the two-step-denaturing and refolding method and the protein refolding screening technique, as well as the continuous dialysis method. First, we identified the refolding buffer composition that allowed for successful refolding to overcome protein precipitation. Next, we used the two-step-denaturing and refolding method and the continuous dialysis method to suppress protein aggregation. In the end, we obtained 15 mg of active MTH1 monomer with 95% purity from 0.5l medium. Integrated refolding techniques proved to be excellent for obtaining the native monomer of S. japonicum MTH1 from inclusion bodies, paving the way for future biological and biophysical studies.

An Automatic Refolding Apparatus for Preparative-Scale Protein Production

Protein refolding is an important process to recover active recombinant proteins from inclusion bodies. Refolding by simple dilution, dialysis and on-column refolding methods are the most common techniques reported in the literature. However, the refolding process is time-consuming and laborious due to the variability of the behavior of each protein and requires a great deal of trial-and-error to achieve success. Hence, there is a need for automation to make the whole process as convenient as possible. In this study, we invented an automatic apparatus that integrated three refolding techniques: varying dilution, dialysis and on-column refolding. We demonstrated the effectiveness of this technology by varying the flow rates of the dilution buffer into the denatured protein and testing different refolding methods. We carried out different refolding methods on this apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12) and thioredoxin fused-human artemin protein (Trx-ARTN); dilution refolding for thioredoxin fused-human insulin-like growth factor I protein (Trx-IGF1) and enhanced fluorescent protein (EGFP); and on-column refolding for bovine serum albumin (BSA). The protein refolding processes of these five proteins were preliminarily optimized using the slowly descending denaturants (or additives) method. Using this strategy of decreasing denaturants concentration, the efficiency of protein refolding was found to produce higher quantities of native protein. The standard refolding apparatus configuration can support different operations for different applications; it is not limited to simple dilution, dialysis and on-column refolding techniques. Refolding by slowly decreasing denaturants concentration, followed by concentration or purification on-column, may be a useful strategy for rapid and efficient recovery of active proteins from inclusion bodies. An automatic refolding apparatus employing this flexible strategy may provide a powerful tool for preparative scale protein production.

SU-E-J-83: CBCT Based Rectum and Bladder Dose Tracking in the Prostate Radiotherapy

Purpose: The aim of this study is to monitor the volume changes of bladder and rectum and evaluate the dosimetric changes of bladder and rectum using daily cone-beam CT for prostate radiotherapy. Methods: The data of this study were obtained from 12 patients, totally 222 CBCTs. All the volume of the bladder and the rectum on the CBCT were normalized to the bladder and the rectum on their own original CT to monitory the volume changes. To evaluate dose delivered to the OARs, volumes that receive 70Gy (V70Gy), 60Gy, 50Gy, 40Gy and 30Gy are calculated for the bladder and the rectum, V20Gy and V10Gy for rectum additionally. And the deviation of the mean dose to the bladder and the rectum are also chosen as the evaluation parameter. Linear regression analysis was performed to identify the mean dose change of the volume change using SPSS 19. Results: The results show that the variances of the normalize volume of the bladder and the rectum are 0.15–0.58 and 0.13–0.50. The variances of V70Gy, V60Gy, V50Gy, V40Gy and V30Gy of bladder are bigger than rectum for 11 patients. The linear regression analysis indicated a negative correlation between the volume and the mean dose of the bladder (p < 0.05). A 10% increase in bladder volume will cause 5.1% (±4.3%) reduction in mean dose. Conclusion: The bladder volume change is more significant than that for rectum for the prostate cancer patient. The volume changes of rectum are not significant except air gap in the rectum. Bladder volume varies will cause significant dose change. The bladder volume monitoring before fractional treatment delivery would be crucial for accuracy dose delivery.