Viral and cellular small integral membrane proteins can modify ion channels endogenous to Xenopus oocytes.

A slowly activated, inward current could be evoked from Xenopus oocytes in response to application of a strong (approximately -190 mV) hyperpolarizing pulse. However, a much lesser hyperpolarization (approximately -130 mV) was able to evoke a similar current from oocytes that expressed the cellular proteins IsK and phospholemman, the synthetic protein SYN-C, and the NB protein of influenza B virus. All of these currents were carried principally by Cl-, and they had similar blocker profiles. The time course (the function of time that described the current increase during a hyperpolarizing voltage-clamp pulse, i.e., activation kinetics) varied from one batch of oocytes to another, but did not vary within each batch with the type of protein expressed. This slowly activated, inward current evoked by hyperpolarization to approximately -130 mV required the expression of a characteristic, minimum level of each of the proteins IsK, SYN-C, and NB. However, not every integral membrane protein expressed in oocytes allowed substantial inward currents to be generated at -130 mV. Oocytes that expressed large amounts of the M2 protein of influenza A virus, which is known to possess an intrinsic cation channel activity, did not display a Cl- current when hyperpolarized to -130 mV. These results suggest that expression of any of the four proteins-IsK, phospholemman, SYN-C, or NB- acts as an activator of an endogenous Cl- conductance.     

The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of nicotiana clevelandii

The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a-green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a-GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a-GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer.     

A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics

Background

The proteomic analysis of body fluids is a growing technology for the identification of protein biomarkers of disease. Given that Papanicolaou tests (Pap tests) are routinely performed on over 30 million women annually in the U.S. to screen for cervical cancer, we examined the residual Pap test fluid as a source of protein for analysis by mass spectrometry (MS). In the liquid-based Pap test, cervical cells are collected from the ectocervix and placed into an alcohol-based fixative prior to staining and pathologic examination. We hypothesized that proteins shed by cells of the female genital tract can be detected in the Pap test fixative by MS-based proteomic techniques. We examined the feasibility of using residual fluid from discarded Pap tests with cytologically “normal” results to optimize sample preparation for MS analysis. The protein composition of the cell-free Pap test fluid was determined by silver staining of sodium dodecyl sulfate -polyacrylamide gels, and the abundance of serum proteins was examined by Western immunoblot using an antibody against human serum albumin. Both pooled and individual samples were trypsin digested and analyzed by two-dimensional MS/MS. Proteins were identified by searching against the Human Uniprot database, and characterized for localization, function and relative abundance.

Results

The average volume of the residual Pap test fluid was 1.5 ml and the average protein concentration was 0.14 mg/ml. By Western immunoblot we showed that the amount of albumin in each sample was significantly reduced compared to normal serum. By MS/MS, we identified 714 unique proteins in pooled Pap test samples and an average of 431 proteins in individual samples. About 40% of the proteins identified were extracellular or localized to the plasma membrane. Almost 20% of the proteins identified were involved in immunity and defense, characteristic of the healthy cervical-vaginal proteome. By merging the protein sets from the individual and pooled Pap test samples, we created a “Normal Pap test Core Proteome” consisting of 153 proteins.

Conclusions

Residual Pap test fluid contains a sufficient amount of protein for analysis by MS and represents a valuable biospecimen source for the identification of protein biomarkers for gynecological diseases.     

Monthly day/night changes and seasonal daily rhythms of sexual steroids in Senegal sole (Solea senegalensis) under natural fluctuating or controlled environmental conditions

In this paper we attempted to investigate the existence of daily fluctuations on plasma sexual steroids (17beta-estradiol, E(2) and testosterone, T) in Senegal sole (Solea senegalensis) females. We described the monthly day/night concentrations and seasonal daily rhythms in animals reared under natural photo- and thermo-period. In addition, the influence of the natural annual fluctuation of the water temperature on the plasma concentration of these steroids was investigated, using one group of Senegal sole under a natural photoperiod, but with an attenuated thermal cycle (around 17-20 degrees C) for one year. Although no significant day/night differences were detected in monthly samplings, the existence of an annual rhythm of E(2) and T (p<0.01) with an acrophase in February was revealed by COSINOR analysis. Maximum values were reached in March for both steroids (6.1+/-1.7 ng mL(-1) at mid-dark, MD and 4.0+/-0.6 ng mL(-1) at mid-light, ML for E2 and 1.4+/-0.4 ng mL(-1) at MD and 0.8+/-0.1 ng mL(-1) at ML for T) in anticipation of the spawning season (May-June). As regards seasonal daily rhythms, the presence of daily oscillations was revealed. At the spring solstice (21st March) a daily rhythm was observed for both steroids (COSINOR, p<0.01), with an acrophase at 20:00 h (E(2)) and at 21:08 h (T). In summer, autumn and winter no daily rhythms were observed due to the low steroid levels at those seasons. When Senegal sole females were submitted to an attenuated annual thermal cycle, the steroid rhythm disappeared (there was no surge in spring, as in the control group) and these fish did not spawn, despite being subjected to natural photoperiod conditions. This result underlined the importance of the natural annual fluctuation of water temperature and photoperiod on the synchronization of the spawning season and on the onset of steroidogenesis.     

Quantitative proteomic analysis by iTRAQ<Superscript>®</Superscript> for the identification of candidate biomarkers in ovarian cancer serum

Background

Ovarian cancer is the most lethal gynecologic malignancy, with the majority of cases diagnosed at an advanced stage when treatments are less successful. Novel serum protein markers are needed to detect ovarian cancer in its earliest stage; when detected early, survival rates are over 90%. The identification of new serum biomarkers is hindered by the presence of a small number of highly abundant proteins that comprise approximately 95% of serum total protein. In this study, we used pooled serum depleted of the most highly abundant proteins to reduce the dynamic range of proteins, and thereby enhance the identification of serum biomarkers using the quantitative proteomic method iTRAQ^®.

Results

Medium and low abundance proteins from 6 serum pools of 10 patients each from women with serous ovarian carcinoma, and 6 non-cancer control pools were labeled with isobaric tags using iTRAQ^® to determine the relative abundance of serum proteins identified by MS. A total of 220 unique proteins were identified and fourteen proteins were elevated in ovarian cancer compared to control serum pools, including several novel candidate ovarian cancer biomarkers: extracellular matrix protein-1, leucine-rich alpha-2 glycoprotein-1, lipopolysaccharide binding protein-1, and proteoglycan-4. Western immunoblotting validated the relative increases in serum protein levels for several of the proteins identified.

Conclusions

This study provides the first analysis of immunodepleted serum in combination with iTRAQ^® to measure relative protein expression in ovarian cancer patients for the pursuit of serum biomarkers. Several candidate biomarkers were identified which warrant further development.

     

Coherence among Different Microbial Source Tracking Markers in a Small Agricultural Stream with or without Livestock Exclusion Practices

Over 1,400 water samples were collected biweekly over 6 years from an intermittent stream protected and unprotected from pasturing cattle. The samples were monitored for host-specific Bacteroidales markers, Cryptosporidium species/genotypes, viruses and coliphages associated with humans or animals, and bacterial zoonotic pathogens. Ruminant Bacteroidales markers did not increase within the restricted cattle access reach of the stream, whereas the ruminant Bacteroidales marker increased significantly in the unrestricted cattle access reach. Human Bacteroidales markers significantly increased downstream of homes where septic issues were documented. Wildlife Bacteroidales markers were detected downstream of the cattle exclusion practice where stream and riparian habitat was protected, but detections decreased after the unrestricted pasture, where the stream and riparian zone was unprotected from livestock. Detection of a large number of human viruses was shown to increase downstream of homes, and similar trends were observed for the human Bacteroidales marker. There was considerable interplay among biomarkers with stream flow, season, and the cattle exclusion practices. There were no to very weak associations with Bacteroidales markers and bacterial, viral, and parasitic pathogens. Overall, discrete sample-by-sample coherence among the different microbial source tracking markers that expressed a similar microbial source was minimal, but spatial trends were physically meaningful in terms of land use (e.g., beneficial management practice) effects on sources of fecal pollution.     

Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation

Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation.Key words: MS-HRM, pyrosequencing, digital PCR, heterogeneous DNA methylation, DAPK1, chronic lymphocytic leukemia     

Detection of human food-borne and zoonotic viruses on irrigated, field-grown strawberries

This study evaluated the presence of pathogenic human and zoonotic viruses on irrigated, field-grown strawberries. Norovirus genogroup I, rotavirus, and swine hepatitis E virus genogroup 3 were detected on strawberries, and irrigation water is suspected as the contamination origin.