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.     

Interspecific diversity reduces and functionally substitutes for intraspecific variation in biofilm communities

Diversity has a key role in the dynamics and resilience of communities and both interspecific (species) and intraspecific (genotypic) diversity can have important effects on community structure and function. However, a critical and unresolved question for understanding the ecology of a community is to what extent these two levels of diversity are functionally substitutable? Here we show, for a mixed-species biofilm community composed of Pseudomonas aeruginosa, P. protegens and Klebsiella pneumoniae, that increased interspecific diversity reduces and functionally substitutes for intraspecific diversity in mediating tolerance to stress. Biofilm populations generated high percentages of genotypic variants, which were largely absent in biofilm communities. Biofilms with either high intra- or interspecific diversity were more tolerant to SDS stress than biofilms with no or low diversity. Unexpectedly, genotypic variants decreased the tolerance of biofilm communities when experimentally introduced into the communities. For example, substituting P. protegens wild type with its genotypic variant within biofilm communities decreased SDS tolerance by twofold, apparently due to perturbation of interspecific interactions. A decrease in variant frequency was also observed when biofilm populations were exposed to cell-free effluents from another species, suggesting that extracellular factors have a role in selection against the appearance of intraspecific variants. This work demonstrates the functional substitution of inter- and intraspecific diversity for an emergent property of biofilms. It also provides a potential explanation for a long-standing paradox in microbiology, in which morphotypic variants are common in laboratory grown biofilm populations, but are rare in diverse, environmental biofilm communities.     

Patient Experience and Satisfaction with Inpatient Service: Development of Short Form Survey Instrument Measuring the Core Aspect of Inpatient Experience

Patient experience reflects quality of care from the patients’ perspective; therefore, patients’ experiences are important data in the evaluation of the quality of health services. The development of an abbreviated, reliable and valid instrument for measuring inpatients’ experience would reflect the key aspect of inpatient care from patients’ perspective as well as facilitate quality improvement by cultivating patient engagement and allow the trends in patient satisfaction and experience to be measured regularly. The study developed a short-form inpatient instrument and tested its ability to capture a core set of inpatients’ experiences. The Hong Kong Inpatient Experience Questionnaire (HKIEQ) was established in 2010; it is an adaptation of the General Inpatient Questionnaire of the Care Quality Commission created by the Picker Institute in United Kingdom. This study used a consensus conference and a cross-sectional validation survey to create and validate a short-form of the Hong Kong Inpatient Experience Questionnaire (SF-HKIEQ). The short-form, the SF-HKIEQ, consisted of 18 items derived from the HKIEQ. The 18 items mainly covered relational aspects of care under four dimensions of the patient’s journey: hospital staff, patient care and treatment, information on leaving the hospital, and overall impression. The SF-HKIEQ had a high degree of face validity, construct validity and internal reliability. The validated SF-HKIEQ reflects the relevant core aspects of inpatients’ experience in a hospital setting. It provides a quick reference tool for quality improvement purposes and a platform that allows both healthcare staff and patients to monitor the quality of hospital care over time.     

Myeloma with involvement of the serous cavities. Cytologic and immunochemical diagnosis and literature review

The significance of serous cavity involvement by myeloma was evaluated in two patients with pleural involvement and two with peritoneal involvement. The involvement occurred at presentation in two patients and after the diagnosis of myeloma in two. The effusions were bloody exudates containing numerous atypical plasma cells. The diagnosis of cavitary involvement was made by morphologic examination of air-dried smears of the effusions, supplemented by the immunocytochemical demonstration of monoclonal proliferation of the plasma cells. In all four cases, these cells contained cytoplasmic kappa light chain immunoglobulins; many of them also stained positively for epithelial membrane antigen. It was best to interpret these immunocytochemical findings with those of the morphologic and additional immunocytochemical studies; the best results for studies for cytoplasmic immunoglobulins were obtained only if the cells in the effusions were washed before they were used for smear preparations and staining. The four patients responded poorly to treatment, dying 12 days, 16 months, 1 month and 10 days after cavitary involvement was recognized. Review of the literature confirmed that the findings in these cases were similar to those in other cases. Cavitary involvement by myeloma carries an ominous prognosis; an accurate recognition of the plasma cells by morphologic and immunocytochemical studies provides the best method of diagnosing cavitary involvement of myeloma and of predicting the poor outcome in such patients.     

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.     

"The orchids have been a splendid sport"--an alternative look at Charles Darwin's contribution to orchid biology

Charles Darwin's work with orchids and his thoughts about them are of great interest and not a little pride for those who are interested in these plants, but they are generally less well known than some of his other studies and ideas. Much has been published on what led to his other books and views. However, there is a paucity of information in the general literature on how Darwin's orchid book came about. This review will describe how The Various Contrivances by Which Orchids Are Fertilised by Insects came into being and will discuss the taxonomy of the orchids he studied. It also will concentrate on some of the less well-known aspects of Darwin's work and observations on orchids-namely, rostellum, seeds and their germination, pollination effects, and resupination-and their influence on subsequent investigators, plant physiology, and orchid science.     

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.     

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.Download video file.^{(145M, mp4)}     

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.