Phylogenomics of five Pseudanabaena cyanophages and evolutionary traces of horizontal gene transfer.

BACKGROUND: Along with the fast development and urbanization in developing countries, the waterbodies aside the growing cities become heavily polluted and highly eutrophic, thus leading to the seasonal outbreak of cyanobacterial bloom. Systematic isolation and characterization of freshwater cyanophages might provide a biological solution to control the awful blooms. However, genomic sequences and related investigations on the freshwater cyanophages remain very limited to date. RESULTS: Following our recently reported five cyanophages Pam1~Pam5 from Lake Chaohu in China, here we isolated another five cyanophages, termed Pan1~Pan5, which infect the cyanobacterium Pseudanabaena sp. Chao 1811. Whole-genome sequencing showed that they all contain a double-stranded DNA genome of 37.2 to 72.0 kb in length, with less than half of the putative open reading frames annotated with known functions. Remarkably, the siphophage Pan1 encodes an auxiliary metabolic gene phoH and constitutes, together with the host, a complete queuosine modification pathway. Proteomic analyses revealed that although Pan1~Pan5 are distinct from each other in evolution, Pan1 and Pan3 are somewhat similar to our previously identified cyanophages Pam3 and Pam1 at the genomic level, respectively. Moreover, phylogenetic analyses suggested that Pan1 resembles the α-proteobacterial phage vB_DshS-R5C, revealing direct evidence for phage-mediated horizontal gene transfer between cyanobacteria and α-proteobacteria. CONCLUSION: In addition to the previous reports of Pam1~Pam5, the present findings on Pan1~Pan5 largely enrich the library of reference freshwater cyanophages. The abundant genomic information provides a pool to identify novel genes and proteins of unknown function. Moreover, we found for the first time the evolutionary traces in the cyanophage that horizontal gene transfer might occur at the level of not only inter-species, but even inter-phylum. It indicates that the bacteriophage or cyanophage could be developed as a powerful tool for gene manipulation among various species or phyla.

Exploration of the genomic landscape of a long-term surviving stage III colorectal cancer patient identifies recurrent and rare mutations: a case report.

A 40-year old female patient from the Chinese Han population presented colorectal cancer (CRC) related symptoms including abdominal discomfort, tenesmus and severe back pain, and was admitted to the First Affiliated Anhui Medical University in October, 2008. The size of her tumor was 3 cm × 3 cm, and the carcinoma had invaded the serosa layer, covering 3/4 of the intestine tube. She was diagnosed with stage III CRC after examination. The patient presented a good prognosis with over 8-year survival after curative surgery and adjuvant therapy with Oxaliplatin and Huaier granules, a traditional Chinese medicine. Using the whole-genome sequencing (WGS) data, we profiled the germline and somatic mutations and obtained an all-inclusive data of the genomic alterations. The genomic alterations were compared with those of stage III CRC patients in The Cancer Genome Atlas Network (TCGA). Mutations in APC, TP53, KRAS, SMAD4, FBXW7 and PIK3CA defined as drivers in TCGA patients were not recorded in our study. However, mutations in MUC4, MUC16, ARID1B, BAZ1A, BRCA2, CTNND1 and NCOA2 rarely reported in TCGA patients were predominant in our patient. Additionally, we observed loss of heterozygosity (LOH) in POLE, RET, BMPR1A, NCOA4 and 30 other genes in contrast to deletion and amplification events recorded in TCGA patients. Overall, we produced a genomic mutation profile of a long-term surviving CRC patient and identified recurrent and rare mutations that could provide a valuable resource for further study into the alterations that characterize advanced CRC which may be useful to design clinical therapy for personalized medicine.

Genome-wide characterization and developmental expression profiling of long non-coding RNAs in Sogatella furcifera.

The genome-wide characterization of long non-coding RNA (lncRNA) in insects demonstrates their importance in fundamental biological processes. Essentially, an in-depth understanding of the functional repertoire of lncRNA in insects is pivotal to insect resources utilization and sustainable pest control. Using a custom bioinformatics pipeline, we identified 1861 lncRNAs encoded by 1852 loci in the Sogatella furcifera genome. We profiled lncRNA expression in different developmental stages and observed that the expression of lncRNAs is more highly temporally restricted compared to protein-coding genes. More up-regulated Sogatella furcifera lncRNA expressed in the embryo, 4th and 5th instars, suggesting that increased lncRNA levels may play a role in these developmental stages. We compared the relationship between the expression of Sogatella furcifera lncRNA and its nearest protein gene and found that lncRNAs were more correlated to their downstream coding neighbors on the opposite strand. Our genome-wide profiling of lncRNAs in Sogatella furcifera identifies exciting candidates for characterization of lncRNAs, and also provides information on lncRNA regulation during insect development.

Integrated analysis of whole genome and transcriptome sequencing in a young patient with gastric cancer provides insights for precision therapy.

Gastric cancer is a leading cause of cancer-associated deaths worldwide and is considered to be an age-related disease. In younger patients, gastric cancer is biologically more aggressive, and prognosis is worse compared with that in elderly patients. In the present case report, the whole genome and transcriptome was sequenced in a 26-year-old patient with gastric cancer who presented with gastric cancer-related symptoms and was admitted to the First Affiliated Anhui Medical Hospital (Hefei, China) in December 2016. In total, 9 germline and 4 somatic mutations were identified in the patient, and there were more deleterious sites in the germline mutated genes. Genes with somatic mutations, such as MUC2, MUC4, SLC8A2, and with structural variations, including CCND3, FGFR2 and FGFR3, were found to be differentially expressed. Cancer-associated pathways, such as the 'calcium signaling pathway', 'cGMP-PKG signaling pathway' and 'transcriptional mis-regulation' were also enriched at both the genomic and transcriptomic levels. The genes found to have germline (SFRP4), somatic (MUC2, MUC4, SLC8A2) mutations, or structural variations (CCND3, FGFR2 and FGFR3) were differentially expressed in the patient and could be promising precision therapy targets.

Genomic Analysis of Mic1 Reveals a Novel Freshwater Long-Tailed Cyanophage.

Lake Chaohu, one of the five largest freshwater lakes in China, has been suffering from severe cyanobacterial blooms in the summer for many years. Cyanophages, the viruses that specifically infect cyanobacteria, play a key role in modulating cyanobacterial population, and thus regulate the emergence and decline of cyanobacterial blooms. Here we report a long-tailed cyanophage isolated from Lake Chaohu, termed Mic1, which specifically infects the cyanobacterium Microcystis aeruginosa. Mic1 has an icosahedral head of 88 nm in diameter and a long flexible tail of 400 nm. It possesses a circular genome of 92,627 bp, which contains 98 putative open reading frames. Genome sequence analysis enabled us to define a novel terminase large subunit that consists of two types of intein, indicating that the genome packaging of Mic1 is under fine control via posttranslational maturation of the terminase. Moreover, phylogenetic analysis suggested Mic1 and mitochondria share a common evolutionary origin of DNA polymerase γ gene. All together, these findings provided a start-point for investigating the co-evolution of cyanophages and its cyanobacterial hosts.

Clinicopathological risk factors for gastric cancer: a retrospective cohort study in China.

OBJECTIVE: To examine the potential clinicopathological factors affecting the prognosis of patients with gastric cancer after surgical treatment in China. METHODS: Between 1 January 2001 and 31 December 2012, a total of 716 patients aged 22-84 years with gastric cancer were enrolled in the study. Survival analysis techniques including log rank test and Cox proportional hazard regression model were applied to evaluate the prognostic significance of clinicopathological characteristics in terms of survival time. RESULTS: Of the 24 demographic and pathological variables collected in the data, 16 prognostic factors of gastric cancer were found to have statistically significant influences on survival time from the unadjusted analyses. The adjusted analysis furtherly revealed that age, age square, lymph node metastasis rate group, tumour size group, surgical type II, number of cancer nodules, invasion depth group and the interaction between surgical type II and tumour size group were important prognosis and clinicopathological factors for gastric cancer in Chinese. CONCLUSION: Our study with relatively large sample size and many potential risk factors enable us to identify independent risk factors associated with the prognosis of gastric cancer. Findings from the current study can be used to assist clinical decision-making, and serve as a benchmark for the planning of future prognosis and therapy for patients with gastric carcinoma.

Identification and characterization of microRNAs in the white-backed planthopper, Sogatella furcifera.

MicroRNAs (miRNAs) are a novel class of small, non-coding endogenous RNAs that play critical regulatory roles in many metabolic activities in eukaryotes. Reports of the identification of miRNAs in Sogatella furcifera (white-backed planthopper), the insect that acts as the only confirmed vector of the southern rice black-streaked dwarf virus (SRBSDV), are limited. In this study, a total of 382 miRNAs were identified in S. furcifera, including 106 conserved and 276 novel miRNAs, using high-throughput sequencing based on two small RNA libraries from viruliferous and non-viruliferous S. furcifera, and these miRNAs belonged to 52 conserved miRNA families and 58 S. furcifera-specific families, respectively. Comparison with miRNAs from 26 insect species and five other species in miRBase showed that more than half of the conserved miRNA families are highly conserved in Hexapoda, while other miRNAs are only conserved in non-dipterans. Furthermore, 4 117 target genes predicted for the 382 identified miRNAs could be categorized into 45 functional groups annotated by Gene Ontology. Compared with non-viruliferous cells, eight up-regulated miRNAs and four down-regulated miRNAs were identified in cells inoculated with SRBSDV, among which miR-14 and miR-n98a may be involved in the immune response to SRBSDV infection. Analyses of the identified miRNAs will provide insights into the roles of these miRNAs in the regulation and expression of genes involved in the metabolism, development and viral infection of S. furcifera.

Evaluation of group A1B erythrocytes converted to type as group O: studies of markers of function and compatibility.

BACKGROUND: Enzymatic conversion of blood group A1B red blood cells (RBC) to group O RBC (ECO) was achieved by combined treatment with α-galactosidase and α-N-acetylgalactosaminidase. The aim of this study was to evaluate the function and safety of these A1B-ECO RBC in vitro. MATERIALS AND METHODS: A 20% packed volume of A1B RBC was treated with enzymes in 250 mM glycine buffer, pH 6.8. The efficiency of the conversion of A and B antigen was evaluated by traditional typing in test tubes, gel column agglutination technology and fluorescence-activated cell sorting (FACS) analysis. The physiological and metabolic parameters of native and ECO RBC were compared, including osmotic fragility, erythrocyte deformation index, levels of 2,3-diphosphoglycerate, ATP, methaemoglobin, free Na(+), and free K(+). The morphology of native and ECO RBC was observed by scanning electron microscopy. Residual α-galactosidase or α-N-acetylgalactosaminidase in A1B-ECO RBC was detected by double-antibody sandwich ELISA method. Manual cross-matching was applied to ensure blood compatibility. RESULTS: The RBC agglutination tests and FACS results showed that A1B RBC were efficiently converted to O RBC. Functional analysis suggested that the conversion process had little impact on the physiological and metabolic parameters of the RBC. The residual amounts of either α-galactosidase or α-N-acetylgalactosaminidase in the A1B-ECO RBC were less than 10 ng/mL of packed RBC. About 18% of group B and 55% of group O sera reacted with the A1B-ECO RBC in a sensitive gel column cross-matching test. DISCUSSION: The conversion process does not appear to affect the morphological, physiological or metabolic parameters of A1B-ECO RBC. However, the A1B-ECO RBC still reacted with some antigens. More research on group O and B sera, which may partly reflect the complexity of group A1 the safety of A1B-ECO RBC is necessary before the application of these RBC in clinical transfusion.

[An introduction of several programs used in genomic analysis].

Genomics is a novel subject that has been developed accompanying with the progress of human genome project. Genomics deals with the chemistry component, structure organization and evolution of genome at global level. As genomics associated with huge data, bioinformatics plays an important role in these processes of data production, data management and data mining. At present, many reliable programs have been used in genomic research successfully, which are usually accessible and downloaded freely. We address here the principles of some programs used wildly in genomics such as sequence alignment, sequence assembly, repeat identification and gene prediction, which are exemplified with typical programs respectively.

[Detection of SARS-CoV RNA in stool samples of SARS patients by nest RT-PCR and its clinical value].

OBJECTIVE: To investigate of severe acute respiratory syndrome (SARS) convalescent stool shedding by RT-PCR. METHODS: One hundred and three stool samples from 46 SARS patients were collected on May 16th, 20th, and 23rd, 2003. For each sample, RNA was extracted using commercial kit and 7 Nest RT-PCR using a 14-pair different SARS-associated coronavirus (SARS-CoV) special primers were carried out simultaneously. RESULTS: Among these 46 SARS patients, 17 cases (37.0%) were stool SARS-CoV RT-PCR negative, and 29 cases (63.0%) were SARS-CoV RT-PCR positive. The duration of positive cases lasted (31.76 +/- 10.78) d (12-64 d). The longest stool shedding case in this study lasted 64 days. Two serial stool samples and for each sample 2 RT-PCR tests using different primers were positive in this case. CONCLUSIONS: Our study observed longest stool shedding of SARS patients to be 64 days after initial onset of SARS. The average stool shedding was 32 days. Hence it is important to think highly of SARS convalescent patient stool sterilization.

[Cultivation of human mesenchymal stem cells on macroporous CultiSpher G microcarriers].

Adult human bone marrow-derived mesenchymal stem cells (MSCs) were cultured on microcarriers in spinner flasks and were compared with those in conventional culture in 12-well plates. For the production of adherently growing MSCs, macroporous CultiSpher G gelatin microcarriers were used in concentration of 1 g/L. The cells were seeded in a density of 5 x 10(4) cells/ml in both spinner culture and conventional stationary culture. The result showed that after 7 days of cultivation a maximum viable cell concentration of 5.15 x 10(5) cells/ml was obtained in spinner culture. Whereas the cell density increased to a maximum of 1.675 x 10(5) cells/ml on day 5 in conventional stationary culture. Lactate was produced up to 12.06 mmol/L in spinner culture and up to 13.10 mmol/L in stationary culture, and glucose was consumed up to 7.38 mmol/L and 5.37 mmol/L respectively. The average lactate yield on glucose consumption in spinner culture was only 1.63, lower than that in stationary culture 2.44. This indicated that the energy metabolism in spinner culture was significantly more efficient than that in conventional culture. After spinner culture for 12 days, the MSCs maintain the characteristics of stem cells. It is concluded that the microcarrier culture system is a suitable way to expand the seeding cells for tissue engineering.

[Single-chain urokinase-type plasminogen activator (scu-PA) purification by immuno-affinity chromatography].

The only difference of primary structure between single-chain prourokinase (pro-UK or scu-PA) and two-chain urokinase (UK or tcu-PA) is the cleavage of a single peptide bond (Lys158-Ile159) and transform scu-PA into its active two-chain form. A 13-peptide (Thr-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile-Gly-Gly-Glu-Cys), which spans the cleavage peptide bond, was synthesized and linked to KLH (Keyhole limpet hemocyanin). The Balb/c mice were immunized by the conjugated protein with proper adjuvant. According to the Kohler and Milstein's methods, a hybridoma cell line G7 secreting monoclonal antibody specific for scu-PA was obtained. The anti-scu-PA McAb, purified from the supernatant of porous microcarrier hybridoma cell culture, was conjugated to CNBr-activated Sepharose 4B to prepare an immuno-affinity chromatography column. The u-PA was purified only by this affinity column from the supernatant of cultivating the u-PA-producing recombinant CHO cell, the u-PA recovery ratio is 90.4%, the purification factor was about 50, with the specific activity of 1.2 x 10(5) IU/mg, the scu-PA ratio in the u-PA product was 96.3%. Compared to immuno-affinity chromatography, the 3-step process for purifying u-PA (cation-exchange column, gel filtration column and benzamidine affinity column) has a u-PA recovery ratio of about 65%, with a specific activity of 1.0 x 10(5) IU/mg, and an scu-PA ratio of about 90%. These results showed that immuno-affinity chromatography is simple to recover u-PA and effective to separate scu-PA from tcu-PA.