The Role of China in the Global Spread of the Current Cholera Pandemic

Epidemics and pandemics of cholera, a severe diarrheal disease, have occurred since the early 19th century and waves of epidemic disease continue today. Cholera epidemics are caused by individual, genetically monomorphic lineages of Vibrio cholerae: the ongoing seventh pandemic, which has spread globally since 1961, is associated with lineage L2 of biotype El Tor. Previous genomic studies of the epidemiology of the seventh pandemic identified three successive sub-lineages within L2, designated waves 1 to 3, which spread globally from the Bay of Bengal on multiple occasions. However, these studies did not include samples from China, which also experienced multiple epidemics of cholera in recent decades. We sequenced the genomes of 71 strains isolated in China between 1961 and 2010, as well as eight from other sources, and compared them with 181 published genomes. The results indicated that outbreaks in China between 1960 and 1990 were associated with wave 1 whereas later outbreaks were associated with wave 2. However, the previously defined waves overlapped temporally, and are an inadequate representation of the shape of the global genealogy. We therefore suggest replacing them by a series of tightly delineated clades. Between 1960 and 1990 multiple such clades were imported into China, underwent further microevolution there and then spread to other countries. China was thus both a sink and source during the pandemic spread of V. cholerae, and needs to be included in reconstructions of the global patterns of spread of cholera.     

DNA Methylation Combinations in Adjacent Normal Colon Tissue Predict Cancer Recurrence: Evidence from a Clinical Cohort Study

Accumulating evidence has suggested the requirement for further stratification of patients in the same tumor stage according to molecular factors. We evaluate the combination of cancer stage and DNA methylation status as an indicator of the risk of recurrence and mortality among patients with colorectal cancer (CRC). A cohort study of 215 patients with CRC (mean age 64.32 years; 50.5% of men) from Tri-Service General Hospital in Taiwan examined the association between cancer stage and risk of CRC recurrence and mortality. A Cox proportional hazard model was used to analyze patient methylation status and clinical information at study entry, and their associations with CRC recurrence and mortality during follow-up. The advanced stage patients with p16, hMLH1, and MGMT methylation were associated with higher risk of CRC recurrence compared with the local stage patients with unmethylation status in tumor tissues, with adjusted hazard ratios (HRs) (95% confidence interval [CI]) of 9.64 (2.92–31.81), 8.29 (3.40–20.22), and 11.83 (3.49–40.12), respectively. When analyzing normal tissues, we observed similar risk of CRC recurrence with adjusted HRs (95% CI) of 10.85 (4.06–28.96), 9.04 (3.79–21.54), and 12.61 (4.90–32.44), respectively. For combined analyses, the risk of recurrence in the patients in advanced stage with DNA methylation in both normal and tumor tissues, compared with local stage with unmethylation, was increased with adjusted HR (95% CI) of 9.37 (3.36–26.09). In the advanced stage patients, methylation status and tissue subtype were associated with increased risk of 5-year cumulative CRC recurrence (p < 0.001). This study demonstrates that clustering DNA methylation status according to cancer stage and tissue subtype is critical for the assessment of risk of recurrence in CRC patients and also indicated an underlying mechanism.     

CPEB4 is a cell survival protein retained in the nucleus upon ischemia or endoplasmic reticulum calcium depletion

The RNA binding protein CPEB (cytoplasmic polyadenylation element binding) regulates cytoplasmic polyadenylation and translation in germ cells and the brain. In neurons, CPEB is detected at postsynaptic sites, as well as in the cell body. The related CPEB3 protein also regulates translation in neurons, albeit probably not through polyadenylation; it, as well as CPEB4, is present in dendrites and the cell body. Here, we show that treatment of neurons with ionotropic glutamate receptor agonists causes CPEB4 to accumulate in the nucleus. All CPEB proteins are nucleus-cytoplasm shuttling proteins that are retained in the nucleus in response to calcium-mediated signaling and alpha-calcium/calmodulin-dependent kinase protein II (CaMKII) activity. CPEB2, -3, and -4 have conserved nuclear export signals that are not present in CPEB. CPEB4 is necessary for cell survival and becomes nuclear in response to focal ischemia in vivo and when cultured neurons are deprived of oxygen and glucose. Further analysis indicates that nuclear accumulation of CPEB4 is controlled by the depletion of calcium from the ER, specifically, through the inositol-1,4,5-triphosphate (IP3) receptor, indicating a communication between these organelles in redistributing proteins between subcellular compartments.     

Construction,expression and characterization of a dual cancer-specific fusion protein targeting carcinoembryonic antigen in intestinal carcinomas

The abundantly expressed carcinoembryonic antigen (CEA) on several cancer types is an attractive target for antibody-directed therapy. However, CEA is also present in some normal tissues. Here, we produced a dual functioning protein, designated as CAtin that exhibits both specific binding and killing functions, by fusing a tumor-specific apoptosis-inducing molecular Apoptin to C-terminus of an anti-CEA single-chain disulfide-stabilized Fv antibody (scdsFv). The CAtin proteins were expressed in Escherichia coli (E. coli), refolded and purified on an immobilized Ni2+ affinity chromatography column. SDS–PAGE and Western blotting revealed that the recombinant protein was well-expressed and the yield was approximately 250 mg/L. We demonstrated by flow cytometry and immunofluorescence assays that CAtin could bind specifically to human colon carcinoma cells (LoVo), but almost not to human uterine cervix (Hela). The results suggest that CAtin is active and specific toward CEA-positive cells and may potentially be used in CEA-targeted cancer therapy.     

Synthesis of 7-benzyl-5-(piperidin-1-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c][2,7]naphthyridin-1-ylamine and its analogs as bombesin receptor subtype-3 agonists

The original structure of a high-throughput screening hit obtained from an external vendor was revised based on multiple NMR studies. The active compound was re-synthesized via a novel route and its structure and biological activity as a BRS-3 agonist were unambiguously confirmed. Multi-gram quantities of the hit were prepared for pharmacokinetic and efficacy studies. The synthetic strategy allowed for the preparation of multiple analogs for SAR exploration.     

Discovery, identification and comparative analysis of non-specific lipid transfer protein (nsLtp) family in Solanaceae

Plant non-specific lipid transfer proteins(nsLtps) have been reported to be involved in plant defense activity against bacterial and fungal pathogens.In this study,we identified 135 (122 putative and 13 previously identified) Solanaceae nsLtps,which are clustered into 8 different groups.By comparing with Boutrot's nsLtp classification,we classified these eight groups into five types (Ⅰ,Ⅱ,Ⅳ,Ⅸ and Ⅹ).We compared Solanaceae nsLtps with Arabidopsis and Gramineae nsLtps and found that (1) Types Ⅰ,Ⅱ and Ⅳ are shared by Solanaceae,Gramineae and Arabidopsis;(2) Types Ⅲ,Ⅴ,Ⅵ and Ⅷ are shared by Gramineae and Arabidopsis but not detected in Solanaceae so far;(3) Type Ⅶ is only found in Gramineae whereas type Ⅸ is present only in Arabidopsis and Solanaceae;(4) Type X is a new type that accounts for 52.59% Solanaceae nsLtps in our data,and has not been reported in any other plant so far.We further built and compared the three-dimensional structures of the eight groups,and found that the major functional diversification within the nsLtp family could be predated to the monocot/dicot divergence,and many gene duplications and sequence variations had happened in the nsLtp family after the monocot/dicot divergence,especially in Solanaceae.     

单核细胞趋化蛋白-1与2型糖尿病大血管病变

单核细胞趋化蛋白-1(monocyte chemoattractant protein-1;MCP-1)属于炎症趋化因子CC亚族成员,它能趋化T淋巴细胞、单核细胞,诱导内皮细胞、单核细胞释放黏附因子,使单核/巨噬细胞向病变处聚集。这些免疫及炎症过程有可能导致2型糖尿病大血管病变的发生、发展。本文就单核细胞趋化蛋白-1促使动脉粥样硬化的机制、及其干预治疗,单核细胞趋化蛋白-1表达上调的影响因素,深入了解单核细胞趋化蛋白-1与2型糖尿病大血管病变的关系。     

Optimization of Pulsed-Field Gel Electrophoresis for Legionella pneumophila Subtyping

A total of 32 strains of Legionella pneumophila were used to optimize pulsed-field gel electrophoresis (PFGE) for subtyping of L. pneumophila. Twenty-six isolates of L. pneumophila with various origins and 11 isolates from five different water systems were used as the panels. For optimization of electrophoretic parameters (EPs) of SfiI PFGE, 26 isolates were analyzed with SfiI digestion, using four EPs yielding the same D value. The EP of a switch time of 5 to 50 s for 21 h had the smallest similarity coefficients and was declared the optimal EP for SfiI PFGE of L. pneumophila. By software analysis and pilot study, AscI was chosen as another PFGE enzyme. AscI PFGE could cluster the isolates from each water system into the same or very similar patterns and had a high degree of typing concordance with other molecular methods. In evaluating the discriminatory power of AscI with the panel of 26 isolates, AscI PFGE gave one single pattern and a D value of 100%. AscI PFGE had a high discriminatory power and a high degree of consistency with epidemiological data and other molecular typing methods for L. pneumophila subtyping, and hence, AscI could be used as a restriction enzyme in PFGE subtyping of L. pneumophila.Legionella pneumophila is an environmental organism that can cause disease in humans and is increasingly recognized as an important pathogen causing nosocomial pneumonia. Potable water systems (14, 26), spa water (28), and cooling towers (7, 13) are among the sources implicated in outbreaks of Legionnaires’ disease. Transmission of bacteria from the environment to humans occurs via inhalation or aspiration of Legionella-containing aerosols (3, 5). Strain differentiation is necessary for the identification of sources of contamination and determination of routes of transmission; this could in turn enable us to more accurately detect outbreaks and limit the spread of L. pneumophila infections. A variety of subtyping techniques have been used to identify and characterize L. pneumophila strains, including monoclonal antibody (MAb) analysis (16, 19), ribotyping (4), amplified fragment length polymorphism (AFLP) analysis (9, 22), PCR-based methods (15, 24), sequence-based typing (SBT) (9, 16), and pulsed-field gel electrophoresis (PFGE) (1, 6).Preliminary reports demonstrated that PFGE is a highly discriminative epidemiological marker for subtyping of L. pneumophila (6, 11, 23, 25), and a number of L. pneumophila PFGE protocols have been described in the literature (1, 2, 4, 14); however, most laboratories that use PFGE to subtype L. pneumophila cannot compare their results because the protocols differ from each other in critical parameters, such as the restriction enzymes and electrophoresis conditions used to generate the DNA fingerprints. To enhance our ability to monitor this pathogen, there is an urgent need for a standardized L. pneumophila PFGE protocol which can readily be implemented in different laboratories for information interpretation.An optimal PFGE protocol produces a suitable number of restriction fragments and gives distinct patterns by agarose gel electrophoresis, with these determined by the restriction enzymes and the electrophoretic parameters (EPs) used. SfiI is the most frequently used enzyme in conventional PFGE protocols for L. pneumophila, and there are several different EPs for SfiI digestion used by investigators for characterization and epidemiological studies. For a certain restriction enzyme, selection of the EP with the smallest similarity coefficients will increase the discriminatory power of PFGE. As the first phase of this study, we compared the similarity coefficients obtained for four EPs with SfiI digestion and determined the one with the maximal discriminatory power.There were some problems found in practical applications of epidemiological investigation of L. pneumophila by PFGE with single SfiI digestion, such as having epidemiologically unrelated strains exhibit the same patterns (30) and the appearance of “ghost” or “phantom” bands. Combination use of two enzymes would give a higher discriminatory power and more accurate results (10, 29). Thus, as the second phase of this study, we selected another suitable enzyme and compared it with SfiI to evaluate the possibility of its use in characterization and epidemiological studies of L. pneumophila.     

Structural characterization of a pectic polysaccharide from Nerium indicum flowers

A polysaccharide fraction, J6, was isolated from the hot-water extract of flowers of oleander Nerium indicum Mill., using ethanol precipitation, cetyltrimethylammonium bromide (CTAB) complexing, anion-exchange chromatography and gel permeation chromatography. J6 was found to contain l-rhamnose, l-arabinose, d-galactose, and d-galacturonic acid, in the ratio of 10.1:49.8:30.1:10.0. Its structure was investigated by methylation analysis, periodate oxidation, Smith degradation, partial acid hydrolysis, electrospray ionization mass spectrometry and NMR spectroscopic methods. It was found that J6 is an RG-I type polysaccharide, which contains a rhamnogalacturonan backbone, with various branches attached to O-4 of l-rhamnose. The branches probably involve (1 → 4)-β-d-galactan, branched l-arabino-(1 → 3)(1 → 6)-β-d-galactan, and (1 → 5)-α-l-arabinan. J6 stimulated NO production of macrophage RAW264.7 cells in a preliminary test.