Investigation into reversed phase chromatography peptide separation systems part II : an evaluation of the robustness of a protocol for column characterisation

The robustness of the Peptide Reversed Phase Chromatography (RPC) Column Characterisation Protocol was evaluated using reduced factorial design, to ascertain the degree of control required for parameters including temperature, flow rate, dwell volume, a systematic shift in the gradient, amount of formic acid in the aqueous and organic, pH of the ammonium formate and amount of acetonitrile (%MeCN) in the strong solvent, where a loss of MeCN resulted in an unacceptable variation. Mitigations have been introduced to ensure the integrity of the data to allow RPC columns to be characterised using peptides as probes, with the definitive protocol described. In addition, the instrument and column batch to batch variability were assessed with good reproducibility

Investigation into reversed phase chromatography peptide separation systems part I : development of a protocol for column characterisation

A protocol was defined which utilised peptides as probes for the characterisation of reversed phase chromatography peptide separation systems. These peptide probes successfully distinguished between differing stationary phases through the probe's hydrophobic, electrostatic, hydrogen bonding and aromatic interactions with the stationary phase, in addition, to more subtle interactions such as the phase's ability to separate racemic or isomeric probes. The dominating forces responsible for the chromatographic selectivity of peptides appear to be hydrophobic as well as electrostatic and polar in nature. This highlights the need for other types of stationary phase ligands with possibly mixed mode functionalities / electrostatic / polar interactions for peptide separations rather than the hydrophobic ligands which dominate small molecule separations. Selectivity differences are observed between phases, but it appears that it is the accessibility differences between these phases which play a crucial role in peptide separations i.e. accessibility to silanols, the hydrophobic acetonitrile / ligand layer or a thin adsorbed water layer on the silica surface

Column classification/characterisation of strong cation exchange phases for the liquid chromatographic analysis of small molecular weight bases

A simple, rapid and robust protocol for the characterisation of strong cation exchange columns for the analysis of small molecular weight bases is described. A range of ten different phases were characterised, and the resultant selectivity and retention factors analysed using Principal Component Analysis. The score plots for the first and second principal components described 83% of the variability within the dataset. Score plots highlighted the large chromatographic differences observed between the phases, the validity of which was established using a larger range of bases. All the strong cation exchange materials demonstrated a synergistic mixed mode (i.e. ion exchange and hydrophobic) retention mechanism. Principal Component Analysis also highlighted the potential difficulty in locating suitable strong cation exchange “back-up” columns for the analysis of small molecular weight bases in that the characterised columns all displayed very different selectivities. The robustness of the protocol was confirmed by a factorial design experiment

Characterization of reversed-phase liquid chromatographic columns containing positively charged functionality

To date, the most commonly used column characterization databases do not determine the relative positive charge associated with new generation RP columns, or they fail to successfully discriminate between RP columns of purportedly low level positive and neutral characters. This paper rectifies this in that it describes a convenient and robust chromatographic procedure for the assessment of the low levels of positive charge on a range of RP columns. The low degree of positive charge was determined by their electrostatic attraction towards the negatively charged 4-n-octylbenzene sulfonic acid (4-OBSA) relative to their retention of the hydrophobic marker toluene (Tol). The new parameter (_α4-OBSA/Tol) was determined for 15 commercially available RP-LC columns. When this was combined with existing Tanaka parameters it was possible to guide the chromatographer towards similar columns as “backup / equivalent phases” or dissimilar columns for exploitation in method development strategies. It should be noted that under certain chromatographic conditions the retention mechanism(s) may be too complex to allow direct location of a “backup / equivalent” column(s). The α4-OBSA/Tol results indicate that even the new generation neutral alkyl phases may exhibit a small degree of positive charge at low buffer concentrations. Mobile phases containing low % MeCN were demonstrated to promote mixed mode (anionic exchange / hydrophobic) retention whereas at high % MeCN anionic exchange retention dominated. The measure of electrostatic repulsion between positively charged columns and positively charged bases was assessed by evaluating the relative retention of a range of bases and neutral analytes. The greatest electrostatic repulsion was observed with hydrophilic bases. While there was no correlation between the positive charge associated with the phases assessed by electrostatic attraction or repulsion, the columns could be broadly divided into three subsets (i.e., significant positive character, medium to low positive character and insignificant positive character). Finally, the results were used to highlight the usefulness of the column characterization database containing the new anionic exchange retention parameter (_α4-OBSA/Tol) for the selection of an equivalent column possessing a low level of positive character in the analysis of a real-life biopharmaceutical application

Predictive validity of the UK clinical aptitude test in the final years of medical school:a prospective cohort study

Peer reviewedPublisher PD

A climatology of clouds in marine cold air outbreaks in both hemispheres

A climatology of clouds within marine cold air outbreaks, primarily using long-term satellite observations, is presented. Cloud properties between cold air outbreaks in different regions in both hemispheres are compared. In all regions marine cold air outbreak clouds tend to be low level with high cloud fraction and low-to-moderate optical thickness. Stronger cold air outbreaks have clouds that are optically thicker, but not geometrically thicker, than those in weaker cold air outbreaks. There is some evidence that clouds deepen and break up over the course of a cold air outbreak event. The top-of-the-atmosphere longwave cloud radiative effect in cold air outbreaks is small because the clouds have low tops. However, their surface longwave cloud radiative effect is considerably larger. The rarity of cold air outbreaks in summer limits their shortwave cloud radiative effect. They do not contribute substantially to global shortwave cloud radiative effect and are, therefore, unlikely to be a major source of shortwave cloud radiative effect errors in climate models

RAB-Like 2 Has an Essential Role in Male Fertility, Sperm Intra-Flagellar Transport, and Tail Assembly

A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2) as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT) proteins and delivers a specific set of effector (cargo) proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein-protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum.This work was supported by grants from the NHMRC to MKO (#606445) and CJO, the Australian Research Council (MKO, RJA, and CJO), the New South Wales Cancer Council (CJO), Cancer Institute New South Wales (CJO), Banque Nationale de Paris-Paribas Australia and New Zealand (CJO), RT Hall Trust (CJO), and the National Breast Cancer Foundation (CJO). JCYL is the recipient of a NHMRC PhD scholarship. MKO and CJO are the recipients of NHMRC Senior Research Fellowships (#545805 and #481310). CCG is the recipient an NHMRC Australia Fellowship. JCW is the recipient of an Australian Research Council Federation Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

DETECTING AND QUANTIFYING REDUCTIVE DECHLORINATION DURING MONITORED NATURAL ATTENUATION AT THE SAVANNAH RIVER CBRP SITE

Various attenuation mechanisms control the destruction, stabilization, and/or removal of contaminants from contaminated subsurface systems. Measuring the rates of the controlling attenuation mechanisms is a key to employing mass balance as a means to evaluate and monitor the expansion, stability and subsequent shrinkage of a contaminant plume. A team of researchers investigated the use of push-pull tests for measuring reductive dechlorination rates in situ at sites with low chlorinated solvent concentrations (<1 ppm). The field research also examined the synergistic use of a suite of geochemical and microbial assays. Previous push-pull tests applied to environmental remediation objectives focused on general hydrological characterization or on designing bioremediation systems by examining the response of the subsurface to stimulation. In this research, the push-pull technique was tested to determine its ''low-range'' sensitivity and uncertainty. Can these tests quantify relatively low attenuation rates representative of natural attenuation? The results of this research indicate that push-pull testing will be useful for measurement of in situ reductive dechlorination rates for chlorinated solvents at ''Monitored Natural Attenuation'' (MNA) sites. Further, using principal component analysis and other techniques, the research confirmed the usefulness of multiple lines of evidence in site characterization and in upscaling measurements made in individual wells--especially for sites where there is a geochemical gradient or varying geochemical regimes within the contaminant plume