Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone λ-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.

Whole-genome multiparametric screening to identify modulators of epithelial-to-mesenchymal transition

Metastasis accounts for the poor prognosis of the majority of solid tumors. The phenotypic transition of nonmotile epithelial tumor cells to migratory and invasive “mesenchymal” cells (epithelial-to-mesenchymal transition [EMT]) enables the transit of cancer cells from the primary tumor to distant sites. There is no single marker of EMT; rather, multiple measures are required to define cell state. Thus, the multiparametric capability of high-content screening is ideally suited for the comprehensive analysis of EMT regulators. The aim of this study was to generate a platform to systematically identify functional modulators of tumor cell plasticity using the bladder cancer cell line TSU-Pr1-B1 as a model system. A platform enabling the quantification of key EMT characteristics, cell morphology and mesenchymal intermediate filament vimentin, was developed using the fluorescent whole-cell-tracking reagent CMFDA and a fluorescent promoter reporter construct, respectively. The functional effect of genome-wide modulation of protein-coding genes and miRNAs coupled with those of a collection of small-molecule kinase inhibitors on EMT was assessed using the Target Activation Bioapplication integrated in the Cellomics ArrayScan platform. Data from each of the three screens were integrated to identify a cohort of targets that were subsequently examined in a validation assay using siRNA duplexes. Identification of established regulators of EMT supports the utility of this screening approach and indicated capacity to identify novel regulators of this plasticity program. Pathway analysis coupled with interrogation of cancer-related expression profile databases and other EMT-related screens provided key evidence to prioritize further experimental investigation into the molecular regulators of EMT in cancer cells.

Identification of eusynstyelamide B as a potent cell cycle inhibitor following the generation and screening of an ascidian-derived extract library using a real time cell analyzer

Ascidians are marine invertebrates that have been a source of numerous cytotoxic compounds. Of the first six marine-derived drugs that made anticancer clinical trials, three originated from ascidian specimens. In order to identify new anti-neoplastic compounds, an ascidian extract library (143 samples) was generated and screened in MDA-MB-231 breast cancer cells using a real-time cell analyzer (RTCA). This resulted in 143 time-dependent cell response profiles (TCRP), which are read-outs of changes to the growth rate, morphology, and adhesive characteristics of the cell culture. Twenty-one extracts affected the TCRP of MDA-MB-231 cells and were further investigated regarding toxicity and specificity, as well as their effects on cell morphology and cell cycle. The results of these studies were used to prioritize extracts for bioassay-guided fractionation, which led to the isolation of the previously identified marine natural product, eusynstyelamide B (1). This bis-indole alkaloid was shown to display an IC50 of 5 μM in MDA-MB-231 cells. Moreover, 1 caused a strong cell cycle arrest in G2/M and induced apoptosis after 72 h treatment, making this molecule an attractive candidate for further mechanism of action studies.

Delirium in advanced cancer : screening for the incidence on admission to an inpatient hospice unit

Background Delirium is a common underdiagnosed condition in advanced cancer leading to increased distress, morbidity, and mortality. Screening improves detection but there is no consensus as to the best screening tool to use with patients with advanced cancer. Objective To determine the incidence of delirium in patients with advanced cancer within 72 hours of admission to an acute inpatient hospice using clinical judgement and validated screening tools. Method One hundred consecutive patients with advanced cancer were invited to be screened for delirium within 72 hours of admission to an acute inpatient hospice unit. Two validated tools were used, the Delirium Rating Scale-Revised 98 (DRS-R-98) and the Confusion Assessment Method (CAM) shortened diagnostic algorithm. These results were compared with clinical assessment by review of medical charts. Results Of 100 consecutive admissions 51 participated and of these 22 (43.1%) screened positive for delirium with CAM and/or DRS-R-98 compared to 15 (29.4%) by clinical assessment. Eleven (21.6%) were identified as hypoactive delirium and 5 (9.8%) as subsyndromal delirium. Conclusion This study confirms that delirium is a common condition in patients with advanced cancer.While there remains a lack of consensus regarding the choice of delirium screening tool this study supports theCAMas being appropriate. Further research may determine the optimal screening tool for delirium enabling the development of best practice clinical guidelines for routinemedical practice.

A simple nutrition screening tool for paediatric inpatients

Background: Pediatric nutrition risk screening tools are not routinely implemented throughout many hospitals, despite prevalence studies demonstrating malnutrition is common in hospitalized children. Existing tools lack the simplicity of those used to assess nutrition risk in the adult population. This study reports the accuracy of a new, quick, and simple pediatric nutrition screening tool (PNST) designed to be used for pediatric inpatients. Materials and Methods: The pediatric Subjective Global Nutrition Assessment (SGNA) and anthropometric measures were used to develop and assess the validity of 4 simple nutrition screening questions comprising the PNST. Participants were pediatric inpatients in 2 tertiary pediatric hospitals and 1 regional hospital. Results: Two affirmative answers to the PNST questions were found to maximize the specificity and sensitivity to the pediatric SGNA and body mass index (BMI) z scores for malnutrition in 295 patients. The PNST identified 37.6% of patients as being at nutrition risk, whereas the pediatric SGNA identified 34.2%. The sensitivity and specificity of the PNST compared with the pediatric SGNA were 77.8% and 82.1%, respectively. The sensitivity of the PNST at detecting patients with a BMI z score of less than -2 was 89.3%, and the specificity was 66.2%. Both the PNST and pediatric SGNA were relatively poor at detecting patients who were stunted or overweight, with the sensitivity and specificity being less than 69%. Conclusion: The PNST provides a sensitive, valid, and simpler alternative to existing pediatric nutrition screening tools such as Screening Tool for the Assessment of Malnutrition in Pediatrics (STAMP), Screening Tool Risk on Nutritional status and Growth (STRONGkids), and Paediatric Yorkhill Malnutrition Score (PYMS) to ensure the early detection of hospitalized children at nutrition risk.

Prognostic validity of 3-Minute Nutrition Screening (3-MinNS) in predicting length of hospital stay, readmission, cost of hospitalisation and mortality : a cohort study

Background: It is important to identify patients who are at risk of malnutrition upon hospital admission as malnutrition results in poor outcomes such as longer length of hospital stay, readmission, hospitalisation cost and mortality. The aim of this study was to determine the prognostic validity of 3-Minute Nutrition Screening (3-MinNS) in predicting hospital outcomes in patients admitted to an acute tertiary hospital through a list of diagnosis-related groups (DRG). Methods: In this study, 818 adult patients were screened for risk of malnutrition using 3-MinNS within 24 hours of admission. Mortality data was collected from the National Registry with other hospitalisation outcomes retrieved from electronic hospital records. The results were adjusted for age, gender and ethnicity, and matched for DRG. Results: Patients identified to be at risk of malnutrition (37%) using 3-MinNS had significant positive association with longer length of hospital stay (6.6 ± 7.1 days vs. 4.5 ± 5.5 days, p<0.001), higher hospitalisation cost (S$4540 ± 7190 vs. S$3630 ± 4961, p<0.001) and increased mortality rate at 1 year (27.8% vs. 3.9%), 2 years (33.8% vs. 7.2%) and 3 years (39.1% vs. 10.5%); p<0.001 for all. Conclusions: The 3-MinNS is able to predict clinical outcomes and can be used to screen newly admitted patients for nutrition risk so that appropriate nutrition assessment and early nutritional intervention can be initiated.

On the need for an international effort to capture, share and use crystallization screening data

When crystallization screening is conducted many outcomes are observed but typically the only trial recorded in the literature is the condition that yielded the crystal(s) used for subsequent diffraction studies. The initial hit that was optimized and the results of all the other trials are lost. These missing results contain information that would be useful for an improved general understanding of crystallization. This paper provides a report of a crystallization data exchange (XDX) workshop organized by several international large-scale crystallization screening laboratories to discuss how this information may be captured and utilized. A group that administers a significant fraction of the worlds crystallization screening results was convened, together with chemical and structural data informaticians and computational scientists who specialize in creating and analysing large disparate data sets. The development of a crystallization ontology for the crystallization community was proposed. This paper (by the attendees of the workshop) provides the thoughts and rationale leading to this conclusion. This is brought to the attention of the wider audience of crystallographers so that they are aware of these early efforts and can contribute to the process going forward. © 2012 International Union of Crystallography All rights reserved.

Dual-functional gum arabic binder for silicon anodes in lithium ion batteries

Si has attracted enormous research and manufacturing attention as an anode material for lithium ion batteries (LIBs) because of its high specific capacity. The lack of a low cost and effective mechanism to prevent the pulverization of Si electrodes during the lithiation/ delithiation process has been a major barrier in the mass production of Si anodes. Naturally abundant gum arabic (GA), composed of polysaccharides and glycoproteins, is applied as a dualfunction binder to address this dilemma. Firstly, the hydroxyl groups of the polysaccharide in GA are crucial in ensuring strong binding to Si. Secondly, similar to the function of fiber in fiberreinforced concrete (FRC), the long chain glycoproteins provide further mechanical tolerance to dramatic volume expansion by Si nanoparticles. The resultant Si anodes present an outstanding capacity of ca. 2000 mAh/g at a 1 C rate and 1000 mAh/g at 2 C rate, respectively, throughout 500 cycles. Excellent long-term stability is demonstrated by the maintenance of 1000 mAh/g specific capacity at 1 C rate for over 1000 cycles. This low cost, naturally abundant and environmentally benign polymer is a promising binder for LIBs in the future.

An efficient forward-backward algorithm to MSDEPP including batteries and voltage control devices

Electric distribution networks are now in the era of transition from passive to active distribution networks with the integration of energy storage devices. Optimal usage of batteries and voltage control devices along with other upgrades in network needs a distribution expansion planning (DEP) considering inter-temporal dependencies of stages. This paper presents an efficient approach for solving multi-stage distribution expansion planning problems (MSDEPP) based on a forward-backward approach considering energy storage devices such as batteries and voltage control devices such as voltage regulators and capacitors. The proposed algorithm is compared with three other techniques including full dynamic, forward fill-in, backward pull-out from the point of view of their precision and their computational efficiency. The simulation results for the IEEE 13 bus network show the proposed pseudo-dynamic forward-backward approach presents good efficiency in precision and time of optimization.