PyroTrimmer: a software with GUI for pre-processing 454 amplicon sequences

The ultimate goal of metagenome research projects is to understand the ecological roles and physiological functions of the microbial communities in a given natural environment. The 454 pyrosequencing platform produces the longest reads among the most widely used next generation sequencing platforms. Since the relatively longer reads of the 454 platform provide more information for identification of microbial sequences, this platform is dedicated to microbial community and population studies. In order to accurately perform the downstream analysis of the 454 multiplex datasets, it is necessary to remove artificially designed sequences located at either ends of individual reads and to correct low-quality sequences. We have developed a program called PyroTrimmer that removes the barcodes, linkers, and primers, trims sequence regions with low quality scores, and filters out low-quality sequence reads. Although these functions have previously been implemented in other programs as well, PyroTrimmer has novelty in terms of the following features: i) more sensitive primer detection using Levenstein distance and global pairwise alignment, ii) the first stand-alone software with a graphic user interface, and iii) various options for trimming and filtering out the low-quality sequence reads. PyroTrimmer, written in JAVA, is compatible with multiple operating systems and can be downloaded free at http://pyrotrimmer.kobic.re.kr.     

MicroRNAome Comparison between Intramuscular and Subcutaneous Vascular Stem Cell Adipogenesis

Background

As an important factor affecting meat quality, intramuscular fat (IMF) content is a topic of worldwide concern. Emerging evidences indicate that microRNAs play important roles in adipocyte differentiation. However, miRNAome has neither been studied during porcine intramuscular preadipocyte differentiation, nor compared with subcutaneous preadipocytes. The objectives of this study were to identify porcine miRNAs involved in adipogenesis in primary preadipocytes, and to determine whether intramuscular and subcutaneous adipocytes differ in the expression and regulation of miRNAs.

Results

miRNAomes in primary intramuscular and subcutaneous adipocytes during differentiation were first sequenced using the Solexa deep sequencing method. The sequences and relative expression levels of 224 known (98.2% in miRbase 18.0) and 280 potential porcine miRNAs were identified. Fifty-four of them changed in similar pattern between intramuscular vascular stem cells (IVSC) and subcutaneous vascular stem cells (SVSC) differentiation, such as miR-210, miR-10b and miR-99a. Expression levels of 10 miRNAs were reversely up-or down-regulated between IVSC and SVSC differentiation, 19 were up-or down-regulated only during IVSC differentiation and 55 only during SVSC differentiation. Additionally, 30 miRNAs showed fat-depot specific expression pattern (24 in cells of intramuscular origin and 6 in cells of subcutaneous origin). These adipogenesis-related miRNAs mainly functioned by targeting similar pathways in adipogenesis, obesity and syndrome.

Conclusion

Comparison of miRNAomes in IVSC and SVSC during differentiation revealed that many different miRNAs are involved in adipogenesis, and they regulate SVSC and IVSC differentiation through similar pathways. These miRNAs may serve as biomarkers or targets for enhancing IMF content, and uncovering their function in IMF development will be of great value in the near future.     

Expression, Purification, and Mass Spectrometric Analysis of (15)N, (13)C-Labeled RGD-Hirudin, Expressed in Pichia pastoris, for NMR Studies

A novel recombinant hirudin, RGD-hirudin, inhibits the activity of thrombin and the aggregation of platelets. Here, we successfully expressed (15)N, (13)C-labeled RGD-hirudin in Pichia pastoris in a fermenter. The protein was subsequently purified to yield sufficient quantities for structural and functional studies. The purified protein was characterized by HPLC and MALDI-TOF mass spectroscopy. Analysis revealed that the protein was pure and uniformly labeled with (15)N and (13)C. A bioassay showed that the anti-thrombin activity and the anti-platelet aggregation ability of the labeled protein were the same as those of unlabeled RGD-hirudin. Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone (15)N, (13)C and (1)H resonance assignments of the r-RGD-Hirudin. The (15)N-(1)H HSQC spectrum of uniformly (15)N, (13)C-labeled RGD-hirudin allowed successful assignment of the signals. Examples of the quality of the data are provided for the (15)N-(l)H correlation spectrum, and by selected planes of the CBCA(CO)NH, CBCANH, and HNCO experiments. These results provide a basis for further studies on the structure-function relationship of RGD-hirudin with thrombin and platelets.     

Navigating the epigenetic landscape of pluripotent stem cells

Pluripotent stem cells, which include embryonic stem cells and induced pluripotent stem cells, use a complex network of genetic and epigenetic pathways to maintain a delicate balance between self-renewal and multilineage differentiation. Recently developed high-throughput genomic tools greatly facilitate the study of epigenetic regulation in pluripotent stem cells. Increasing evidence suggests the existence of extensive crosstalk among epigenetic pathways that modify DNA, histones and nucleosomes. Novel methods of mapping higher-order chromatin structure and chromatin-nuclear matrix interactions also provide the first insight into the three-dimensional organization of the genome and a framework in which existing genomic data of epigenetic regulation can be integrated to discover new rules of gene regulation.     

Identification and expansion of cancer stem cells in tumor tissues and peripheral blood derived from gastric adenocarcinoma patients

Gastric cancer is the fourth most common cancer worldwide, with a high rate of death and low 5-year survival rate. To date, there is a lack of efficient therapeutic protocols for gastric cancer. Recent studies suggest that cancer stem cells (CSCs) are responsible for tumor initiation, invasion, metastasis, and resistance to anticancer therapies. Thus, therapies that target gastric CSCs are attractive. However, CSCs in human gastric adenocarcinoma (GAC) have not been described. Here, we identify CSCs in tumor tissues and peripheral blood from GAC patients. CSCs of human GAC (GCSCs) that are isolated from tumor tissues and peripheral blood of patients carried CD44 and CD54 surface markers, generated tumors that highly resemble the original human tumors when injected into immunodeficient mice, differentiated into gastric epithelial cells in vitro, and self-renewed in vivo and in vitro. Our findings suggest that effective therapeutic protocols must target GCSCs. The capture of GCSCs from the circulation of GAC patients also shows great potential for identification of a critical cell population potentially responsible for tumor metastasis, and provides an effective protocol for early diagnosis and longitudinal monitoring of gastric cancer.     

Dry-heat treatment process for enhancing viral safety of an antihemophilic factor VIII concentrate prepared from human plasma

Viral safety is a prerequisite for manufacturing clinical antihemophilic factor VIII concentrates from human plasma. With particular regard to the hepatitis A virus (HAV), a terminal dry-heat treatment (100 degrees for 30 min) process, following lyophilization, was developed to improve the virus safety of a solvent/detergent-treated antihemophilic factor VIII concentrate. The loss of factor VIII activity during dry-heat treatment was of about 5%. No substantial changes were observed in the physical and biochemical characteristics of the dry-heat-treated factor VIII compared with those of the factor VIII before dry-heat treatment. The dry-heat-treated factor VIII was stable for up to 24 months at 4oC. The dry-heat treatment after lyophilization was an effective process for inactivating viruses. The HAV, murine encephalomyocarditis virus (EMCV), and human immunodeficiency virus (HIV) were completely inactivated to below detectable levels within 10 min of the dry-heat treatment. Bovine herpes virus (BHV) and bovine viral diarrhea virus (BVDV) were potentially sensitive to the treatment. However porcine parvovirus (PPV) was slightly resistant to the treatment. The log reduction factors achieved during lyophilization and dry-heat treatment were > or =5.55 for HAV, > or =5.87 for EMCV, > or =5.15 for HIV, 6.13 for BHV, 4.46 for BVDV, and 1.90 for PPV. These results indicate that dry-heat treatment improves the virus safety of factor VIII concentrates, without destroying the activity. Moreover, the treatment represents an effective measure for the inactivation of non-lipid-enveloped viruses, in particular HAV, which is resistant to solvent/detergent treatment.     

Free radicals run in lizard families

In the ageing individual, the production of reactive oxygen species (ROS) accelerates with cell senescence. Depending on the heritability of the underlying processes that determine net ROS levels, this may influence ageing per se and its evolutionary direction and rate of change. In order to understand the inheritance and evolution of net ROS levels in free-ranging lizards, we used flow cytometry together with ROS-sensitive fluorogenic probes to measure ROS in lizard blood cells. We measured basal levels of (i) non-specific ROS (superoxide, singlet oxygen, H2O2 and peroxynitrite), (ii) superoxide specifically and (iii) superoxide after CCCP treatment, which elevated ROS production in the mitochondria. The cumulative level of non-specific ROS was higher in adults than juveniles and superoxide level showed high heritability and variability among families. We suggest that the evolution of ROS dynamics may be ROS species specific and perhaps depend on the relative degree of uni- or biparental inheritance of ROS main regulatory pathways.