Construction of photodynamic‐effect immunofluorescence probes by a complex of quantum dots,immunoglobulin G and chlorin e6 and their application in HepG2 cell killing

In this study, tri‐functional immunofluorescent probes (Ce6–IgG–QDs) based on covalent combinations of quantum dots (QDs), immunoglobulin G (IgG) and chlorin e6 (Ce6) were developed and their photodynamic ability to induce the death of cancer cells was demonstrated. Strategically, one type of second‐generation photosensitizer, Ce6, was first coupled with anti‐IgG antibody using the EDC/NHS cross‐linking method to construct the photosensitive immunoconjugate Ce6–IgG. Then, a complex of Ce6–IgG–QDs immunofluorescent probes was obtained in succession by covalently coupling Ce6–IgG to water soluble CdTe QDs. The as‐manufactured Ce6–IgG–QDs maintained the bio‐activities of both the antigen–antibody‐based tumour targeting effects of IgG and the photodynamic‐related anticancer activities of Ce6. By way of polyclonal antibody interaction with rabbit anti‐human epidermal growth factor receptor (anti‐EGFR antibody, N‐terminus), Ce6–IgG–QDs were labelled indirectly onto the surface of human hepatocarcinoma (HepG2) cells in cell recognition and killing experiments. The results indicated that the Ce6–IgG–QDs probes have excellent tumour cell selectivity and higher photosensitivity in photodynamic therapy (PDT) compared with Ce6 alone, due to their antibody‐based specific recognition and location of HepG2 cells and the photodynamic effects of Ce6 killed cells based on efficient fluorescence resonance energy transfer between QDs and Ce6. Copyright © 2015 John Wiley & Sons, Ltd.     

miR-29a attenuates cardiac hypertrophy through inhibition of PPARδ expression

Although cardiac hypertrophy is widely recognized as a risk factor that leads to cardiac dysfunction and, ultimately, heart failure, the complex mechanisms underlying cardiac hypertrophy remain incompletely characterized. The nuclear receptor peroxisome proliferator-activated receptor δ (PPARδ) is involved in the regulation of cardiac lipid metabolism. Here, we describe a novel PPARδ-dependent molecular cascade involving microRNA-29a (miR-29a) and atrial natriuretic factor (ANF), which is reactivated in cardiac hypertrophy. In addition, we identify a novel role of miR-29a, in which it has a cardioprotective function in isoproterenol hydrochloride-induced cardiac hypertrophy by targeting PPARδ and downregulating ANF. Finally, we provide evidence that miR-29a reduces the isoproterenol hydrochloride-induced cardiac hypertrophy response, thereby underlining the potential clinical relevance of miR-29a in which it may serve as a potent therapeutic target for heart hypertrophy treatment.     

Genome-wide analysis of codon usage bias patterns in an enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> F18 strain

Enterogenic Escherichia coli (ETEC) F18 strains are the main pathogenic bacteria causing severe diarrhea in humans and domestic animals. However, the information about synonymous codon usage pattern of ETEC F18 genome remains unclear. We conducted a genome-wide analysis of synonymous codon usage patterns in the ETEC F18 strain SRA: SAMN02471895. After filtering of the complete genome sequence, 4327 coding sequences were analyzed using multivariate statistical methods to calculate synonymous codon usage patterns and to evaluate the influence of various factors in shaping the codon usage. The mean GC content was 51.38%, with a slight preference for G/C-ending codons. Twenty-two codons were determined as ‘‘optimal codons”. ENC plots showed some of the genes were on or close to the expected curve, while only points with low-ENC values were below the curve. PR2 analysis showed that GC and AT were not used proportionally, suggesting major roles for mutational pressure and natural selection in shaping usage. Neutrality plots showed a significant correlation between GC12 and GC3, suggesting that mutational pressure is responsible for nucleotide composition in shaping the strength of codon usage. Translational selection was the main factor shaping the codon usage pattern of ETEC F18 genome, while other factors such as protein length, GRAVY and ARO values also influenced codon usage to some extent. We analyzed the codon usage pattern systematically and identified the factors shaping codon usage bias in the ETEC F18 genome. Such information further elucidates the mechanisms of synonymous codon usage bias and provides the basis of molecular genetic engineering and evolutionary studies.     

Genetic analysis of Canarium album in different areas of China by improved RAPD and ISSR

To lay the foundation of the classification of Canarium album (C. album), and C. album from Terminalia Chebula (T. chebula) in different areas of China, improved RAPD and ISSR analysis were performed to analyze polymorphism and genetic relationship. Ten samples were collected from different locations in China. A total of 221 fragments were detected by improved RAPD, out of which polymorphic fragments accounted for 82.3% with average amplification bands of 10.05 per primer. ISSR markers revealed a total of 147 alleles, where polymorphic fragments accounted for 83.5%, with average amplification bands of 7.35 per primer. The sizes of fragments ranged from 200 to 2500 bp and from 150 to 2000 bp in RAPD and ISSR markers, respectively. The similarity coefficient ranged from 0.46 to 0.86 with RAPD markers and 0.36 to 0.89 with ISSR markers. The results indicated that improved RAPD and ISSR methods are useful for genetic diversity study of C. album. Thus, this study provides us the theoretical basis for the breeding and classification of C. album in South and Southwest China.     

Preparation and analysis of spermatocyte meiotic pachytene bivalents of pigs for gene mapping

Well-spread meiotic pachytene bivalents were obtained by using the prolonged hypotonic treatment combined with high chloroform Carnory's fixative solution from cells of the testes of domestic pigs. Comparison in the division index and length of pachytene bivalents with metaphase chromosomes showed that those of the former are 5 times higher and 3.42(1.87-5.98) times longer than those of the latter. Comparative studies on chromomere maps of bivalents and mitotic chromosomal G-bands were conducted by using the chromosome 12 as a example. Sex vesicle and various shapes of synaptic sex chromosomes have been observed. Two-color PRimed IN Situ (PRINS) labeling has been conducted successfully on pachytene bivalents of pigs.     

Isolation of transcription factors binding auxin response elements using a yeast one-hybrid system

Plant hormones play an important role during higher plant embryogenesis. Auxin is central to the development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses. Auxin response element (AuxRE), present in the promoters of many auxin-induced genes, can confer auxin responsiveness. Using carrot somatic embryo under specific developmental phase, a cDNA expression library was constructed. Several plasmids were recombined containing the tetramer of AuxRE as a bait. After screening by a yeast one-hybrid system, one positive clone was confirmed and characterized. Electrophoretic mobility shift assay showed that AxRF1 protein expressed in yeast cell could bind AuxRE in vitro. It suggests that AxRF1 participates in regulation of the expression of auxin responsive gene during carrot somatic embryogenesis.     

Cloning of two cDNAs encoding a family of ATP sulfurylase from Camellia sinensis related to selenium or sulfur metabolism and functional expression in Escherichia coli.

ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of two cDNAs encoding ATP sulfurylase (APS1 and APS2) from Camellia sinensis. They were isolated by RT-PCR and RACE-PCR reactions. The expression of APS1 and APS2 are correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1415-bp cDNA with an open reading frame predicted to encode a 360-amino acid, 40.5kD protein; APS2 is a 1706-bp cDNA with an open reading frame to encode a 465-amino acid, 51.8kD protein. The predicted amino acid sequences of APS1 and APS2 have high similarity to ATP sulfurylases of Medicago truncatula and Solanum tuberosum, with 86% and 84% identity respectively. However, they share only 59.6% identity with each other. The enzyme extracts prepared from recombinant Escherichia coli containing Camellia sinensis APS genes had significant enzyme activity.     

Genome sequence of duck pathogen Mycoplasma anatis strain 1340

Mycoplasma anatis, a member of the class Mollicutes, is the causative agent of a contagious infectious disease of domestic ducklings, wild birds, and eggs. Increasing reports show that coinfection of M. anatis with Escherichia coli results in substantial economic impacts on the duck farms in China. Here, we announce the first genome sequence of M. anatis.     

Determination of an Optimal Standardized Uptake Value of Fluorodeoxyglucose for Positron Emission Tomography Imaging to Assess Pathological Volumes of Cervical Cancer: A Prospective Study

Purpose

To determine the optimal standardized uptake value (SUV) of ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) for positron emission tomography (PET) imaging, at which the PET-defined gross tumor volume (GTV_PET) best matches with the pathological volume (GTV_PATH) in the cervical cancer.

Materials and Methods

Ten patients with the cervical cancer who underwent surgery were enrolled in this study. The excised specimens were processed for whole-mount serial sections and H-E staining. The tumor borders were outlined in sections under a microscope, histopathological images were scanned and the GTV_PATH calculated. The GTV_PET was delineated automatically by using various percentages relative to the maximal SUV and absolute SUV. The optimal threshold SUV was further obtained as the value at which the GTV_PET best matched with the GTV_PATH.

Results

An average of 85±10% shrinkage of tissue was observed after the formalin fixation. The GTV_PATH was 13.38±2.80 cm³ on average. The optimal threshold on percentile SUV and absolute SUV were 40.50%±3.16% and 7.45±1.10, respectively. The correlation analysis showed that the optimal percentile SUV threshold was inversely correlated with GTV_PATH (p<0.05) and tumor diameter (p<0.05). The absolute SUV was also positively correlated with SUV_max (p<0.05).

Conclusion

The pathological volume could provide the more accurate tumor volume. The optimal SUV of FDG for PET imaging by use of GTV_PATH as standard for cervical cancer target volume delineation was thus determined in this study, and more cases are being evaluated to substantiate this conclusion.