A new genotyping method for detecting low abundance single nucleotide mutations based on gap ligase chain reaction and quantitative PCR assay

We tested applicability of a new genotyping technique to detect a low abundance CD17 (A → T) mutation of β-globin gene. The technique utilized a combined gap ligase chain reaction (Gap-LCR) and quantitative PCR (qPCR) methods. One pair of Gap-LCR primers was modified by adding specific sequences to the 5′ end of the upstream and the 3′ end of the downstream primer which served as a combining sequence for qPCR. First, specific mutation is detected using Gap-LCR; then, ligation products are detected by qPCR. Our results show that the amount of LCR products is directly proportional to the amount of template DNA. We further demonstrate that this technique detects a low abundance mutant DNA with a mutant/normal allele ratio as low as 1:10000. This technique was applied to detect a paternally inherited CD17 mutation from 53 maternal plasma samples. The results were consistent with those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. In conclusion, by combining Gap-LCR and qPCR technology we successfully established a highly sensitive technique to detect low abundance point mutations. This technique can be applied to detect fetal DNA point mutation in maternal plasma.     

Intratumoral heterogeneity determines discordant results of diagnostic tests for human epidermal growth factor receptor (HER) 2 in gastric cancer specimens

Accurate assessment of human epidermal growth factor receptor (HER) 2 is essential for efficient selection of patients who may benefit from therapies targeting this surface receptor (e.g., trastuzumab). Intratumoral heterogeneity of HER2 expression may potentially contribute to inaccurate assessment of HER2 status. To clarify intratumoral heterogeneity of HER2 expression and its potential clinical impact on assessment of HER2 status, we analyzed 148 endoscopic biopsy specimens and 117 excisional tumor specimens collected from 148 patients with primary gastric cancer. Specifically, we assessed HER2 protein overexpression and gene amplification using, respectively, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). There were 28 IHC-positive cases and 25 FISH-positive cases among these 148 patients. Heterogeneous HER2 protein expression was demonstrated in 23 of 29 (79.3%) IHC-positive cases, while gene expression heterogeneity was found in 11 of 25 (44.0%) FISH-positive cases. Intratumoral heterogeneity was the main reason of discordant results between IHC and FISH or between endoscopic biopsy and excisional tumor specimens. The clinical significance and impact of intratumoral HER2 expression heterogeneity on treatment outcome in gastric cancer require further studies.     

High-throughput screening for anti-influenza A virus drugs and study of the mechanism of procyanidin on influenza A virus-induced autophagy

In this research, we have established a high-throughput screening (HTS) platform based on the influenza A virus (IAV) vRNA promoter. Using this HTS platform, we selected 35 medicinal plants out of 83 examples of traditional Chinese medicine and found that 7 examples had not been reported. After examining many previous reports, we found that Vaccinium angustifolium Ait., Vitis vinifera L, and Cinnamomum cassia Presl had a common active compound, procyanidin, and then determined the anti-IAV effect of procyanidin and explored its mechanism of action. With a plaque inhibition assay and a time-of-addition experiment, we found that procyanidin could inhibit the IAV replication at several stages of the life cycle. In the Western blot and EGFP-LC3 localization assays, we found that procyanidin could inhibit the accumulation of LC3II and the dot-like aggregation of EGFP-LC3. In the RT-PCR and Western blot assays, we found procyanidin could inhibit the expression of Atg7, Atg5, and Atg12. Finally, by the bimolecular fluorescence complementation-fluorescence resonance energy transfer and co-immunoprecipitation assays, we found that procyanidin could inhibit the formation of the Atg5-Atg12/Atg16 heterotrimer and the dissociation of the beclin1/bcl2 heterodimer. In conclusion, we have established an HTS platform and identified procyanidin as a novel and promising anti-IAV agent.     

In vitro development and transfer of resistance to chlortetracycline in Bacillus subtilis

The present criteria and rules controlling the approval of the use of probiotics are limited to antibiotic resistance patterns and the presence of antibiotic resistance genes in bacteria. There is little information available in the literature regarding the risk of the usage of probiotics in the presence of antibiotic pressure. In this study we investigated the development and transfer of antibiotic resistance in Bacillus subtilis selected in vitro by chlortetracycline in a stepwise manner. Bacillus subtilis was exposed to increasing concentrations of chlortetracyclineto induce in vitro resistance to chlortetracycline, and the minimal inhibitory concentrations were determinedfor the mutants. Resistant B. subtilis were conjugated with Escherichia coli NK5449 and Enterococcus faecalis JH2-2 using the filter mating. Three B. subtilis tetracycline resistant mutants (namely, BS-1, BS-2, and BS-3) were derived in vitro. A tetracycline resistant gene, tet (K), was found in the plasmids of BS-1 and BS-2. Three conjugates (BS-1N, BS-2N, and BS-3N) were obtained when the resistant B. subtilis was conjugated with E. coli NK5449. The conjugation frequencies for the BS-1N, BS-2N, and BS-3N conjugates were 4.57×10^?7, 1.4×10^?7, and 1.3×10^?8, respectively. The tet(K) gene was found only in the plasmids of BS-1N. These results indicate that long-term use of probiotics under antibiotic selection pressure could cause antibiotic resistance, and the resistance gene could be transferred to other bacteria. The risk arising from the use of probiotics under antibiotic pressure should be considered in the criteria and rules for the safety assessment of probiotics.     

Characterization of the Hormone and Stress-Induced Expression of <Emphasis Type="Italic">FaRE</Emphasis>1 Retrotransposon Promoter in Strawberry

Retrotransposons are the most abundant mobile elements in the plant genome and seem to play an important role in genome reorganization induced by environmental challenges. Their success in this function depends on the ability of their promoters to regulate plant adaptation to biotic and abiotic stresses. In this study, the promoter region of FaRE1 was amplified in the strawberry genome, and promoter::GUS fusion was constructed. We produced transgenic strawberry plants carrying FaRE1 promoter::GUS-fusion genes, and monitored GUS reporter activity. Histochemical and fluorimetric GUS analysis these plants showed the characteristics of the FaRE1 promoter were activated by either hormones treatments with ABA, NAA, and 2,4-D or cold stress. In addition, we found the GUS reporter was activated in the leaves of transgenic strawberry plants using 5-azaC. These results suggest that the promoter of FaRE1 may act as different signal transduction pathways, allowing FaRE1 retrotransposon to be activated in response to multiples challenges.     

A Novel Immunodiagnostic Assay to Detect Serum Antibody Response against Selected Soluble Egg Antigen Fractions from Schistosoma japonicum

Background

Schistosomiasis japonica remains a real threat to public health in China. The currently used immunodiagnostic assays are sensitive and have a certain degree of specificity, however, they all use complex crude antigens, are based on detection of schistosome-specific antibodies, and have been shown to cross-react with other parasitic diseases. Therefore, these assays cannot be used to evaluate chemotherapy efficacy. The development of highly sensitive and highly specific immunodiagnostic techniques that can monitor the decline of antibodies specific for S. japonica will be extremely valuable as part of the ongoing strategy to control schistosomiasis in endemic areas. Here we report on the identification of unique fraction antigens of soluble egg antigen (SEA) to which the antibodies disappear 7 weeks after effective treatment. Furthermore, we use these SEA fractions to develop a modified assay with both high sensitivity and specificity.

Methodology/Principal Findings

SEA of S. japonicum was fractionated by electrophoresis using 7.5% SDS-PAGE under non-reducing conditions. The SEA fraction antigens to which antibodies were decreased soon after treatment were collected and used as the detection antigens to establish the FA-ELISA. Sera from patients with acute and chronic schistosomiasis infection, healthy people, and those with other parasitic diseases, were used to evaluate their sensitivity and specificity. Furthermore, sera from patients with chronic schistosomiasis infection were evaluated before and after treatment at different time points to evaluate their chemotherapeutic efficacy.

Conclusion/Significance

We demonstrated that this novel FA-ELISA provided high sensitivity and specificity, with very low cross-reactivity, and can serve as an effective tool to determine the efficacy of chemotherapy against S. japonicum.     

Analysis of autophagy genes in microalgae: chlorella as a potential model to study mechanism of autophagy

Background

Microalgae, with the ability to mitigate CO₂ emission and produce carbohydrates and lipids, are considered one of the most promising resources for producing bioenergy. Recently, we discovered that autophagy plays a critical role in the metabolism of photosynthetic system and lipids production. So far, more than 30-autophagy related (ATG) genes in all subtypes of autophagy have been identified. However, compared with yeast and mammals, in silico and experimental research of autophagy pathways in microalgae remained limited and fragmentary.

Principal Findings

In this article, we performed a genome-wide analysis of ATG genes in 7 microalgae species and explored their distributions, domain structures and evolution. Eighteen “core autophagy machinery” proteins, four mammalian-specific ATG proteins and more than 30 additional proteins (including “receptor-adaptor” complexes) in all subtypes of autophagy were analyzed. Data revealed that receptor proteins in cytoplasm-to-vacuole targeting and mitophagy seem to be absent in microalgae. However, most of the “core autophagy machinery” and mammalian-specific proteins are conserved among microalgae, except for the ATG9-cycling system in Chlamydomonas reinhardtii and the second ubiquitin-like protein conjugation complex in several algal species. The catalytic and binding residues in ATG3, ATG5, ATG7, ATG8, ATG10 and ATG12 are also conserved and the phylogenetic tree of ATG8 coincides well with the phylogenies. Chlorella contains the entire set of the core autophagy machinery. In addition, RT-PCR analysis verified that all crucial ATG genes tested are expressed during autophagy in both Chlorella and Chlamydomonas reinhardtii. Finally, we discovered that addition of 3-Methyladenine (a PI3K specific inhibitor) could suppress the formation of autophagic vacuoles in Chlorella.

Conclusions

Taken together, Chlorella may represent a potential model organism to investigate autophagy pathways in photosynthetic eukaryotes. The study will not only promote understanding of the general features of autophagic pathways, but also benefit the production of Chlorella-derived biofuel with future commercial applications.     

Metagenomic insights into the fibrolytic microbiome in yak rumen

The rumen hosts one of the most efficient microbial systems for degrading plant cell walls, yet the predominant cellulolytic proteins and fibrolytic mechanism(s) remain elusive. Here we investigated the cellulolytic microbiome of the yak rumen by using a combination of metagenome-based and bacterial artificial chromosome (BAC)-based functional screening approaches. Totally 223 fibrolytic BAC clones were pyrosequenced and 10,070 ORFs were identified. Among them 150 were annotated as the glycoside hydrolase (GH) genes for fibrolytic proteins, and the majority (69%) of them were clustered or linked with genes encoding related functions. Among the 35 fibrolytic contigs of >10 Kb in length, 25 were derived from Bacteroidetes and four from Firmicutes. Coverage analysis indicated that the fibrolytic genes on most Bacteroidetes-contigs were abundantly represented in the metagenomic sequences, and they were frequently linked with genes encoding SusC/SusD-type outer-membrane proteins. GH5, GH9, and GH10 cellulase/hemicellulase genes were predominant, but no GH48 exocellulase gene was found. Most (85%) of the cellulase and hemicellulase proteins possessed a signal peptide; only a few carried carbohydrate-binding modules, and no cellulosomal domains were detected. These findings suggest that the SucC/SucD-involving mechanism, instead of one based on cellulosomes or the free-enzyme system, serves a major role in lignocellulose degradation in yak rumen. Genes encoding an endoglucanase of a novel GH5 subfamily occurred frequently in the metagenome, and the recombinant proteins encoded by the genes displayed moderate Avicelase in addition to endoglucanase activities, suggesting their important contribution to lignocellulose degradation in the exocellulase-scarce rumen.