Choice of antibody is critical for specific and sensitive detection of androgen receptor splice variant-7 in circulating tumor cells

Androgen receptor variant 7 (AR-V7) is an important biomarker to guide treatment options for castration-resistant prostate cancer (CRPC) patients. Its detectability in circulating tumour cells (CTCs) opens non-invasive diagnostic avenues. While detectable at the transcript level, AR-V7 protein detection in CTCs may add additional information and clinical relevance. The aim of this study was to compare commercially available anti-AR-V7 antibodies and establish reliable AR-V7 immunocytostaining applicable to CTCs from prostate cancer (PCa) patients. We compared seven AR-V7 antibodies by western blotting and immmunocytostaining using a set of PCa cell lines with known AR/AR-V7 status. The emerging best antibody was validated for detection of CRPC patient CTCs enriched by negative depletion of leucocytes. The anti-AR-V7 antibody, clone E308L emerged as the best antibody in regard to signal to noise ratio with a specific nuclear signal. Moreover, this antibody detects CRPC CTCs more efficiently compared to an antibody previously shown to detect AR-V7 CTCs. We have determined the best antibody for AR-V7 detection of CTCs, which will open future studies to correlate AR-V7 subcellular localization and potential co-localization with other proteins and cellular structures to patient outcomes

Distinct microbiota dysbiosis in patients with non-erosive reflux disease and esophageal adenocarcinoma

Non-erosive reflux disease (NERD) and esophageal adenocarcinoma (EAC) are often regarded as bookends in the gastroesophageal reflux disease spectrum. However, there is limited clinical evidence to support this disease paradigm while the underlying mechanisms of disease progression remain unclear. In this study, we used 16S rRNA sequencing and mass-spectrometer-based proteomics to characterize the esophageal microbiota and host mucosa proteome, respectively. A total of 70 participants from four patient groups (NERD, reflux esophagitis, Barrett’s esophagus, and EAC) and a control group were analyzed. Our results showed a unique NERD microbiota composition, distinct to control and other groups. We speculate that an increase in sulfate-reducing Proteobacteria and Bacteroidetes along with hydrogen producer Dorea are associated with a mechanistic role in visceral hypersensitivity. We also observed a distinct EAC microbiota consisting of a high abundance of lactic acid-producing bacteria (Staphylococcus, Lactobacillus, Bifidobacterium, and Streptococcus), which may contribute towards carcinogenesis through dysregulated lactate metabolism. This study suggests the close relationship between esophageal mucosal microbiota and the appearance of pathologies of this organ

Human Group IIA Phospholipase A2 : three decades on from its discovery

Phospholipase A2 (PLA2) enzymes were first recognized as an enzyme activity class in 1961. The secreted (sPLA2) enzymes were the first of the five major classes of human PLA2s to be identified and now number nine catalytically-active structurally homologous proteins. The best-studied of these, group IIA sPLA2, has a clear role in the physiological response to infection and minor injury and acts as an amplifier of pathological inflammation. The enzyme has been a target for anti-inflammatory drug development in multiple disorders where chronic inflammation is a driver of pathology since its cloning in 1989. Despite intensive effort, no clinically approved medicines targeting the enzyme activity have yet been developed. This review catalogues the major discoveries in the human group IIA sPLA2 field, focusing on features of enzyme function that may explain this lack of success and discusses future research that may assist in realizing the potential benefit of targeting this enzyme. Functionally-selective inhibitors together with isoform-selective inhibitors are necessary to limit the apparent toxicity of previous drugs. There is also a need to define the relevance of the catalytic function of hGIIA to human inflammatory pathology relative to its recently-discovered catalysis-independent function

The first accurate measurement of somatostatin concentration in human blood

The primary structure of the cyclic peptide which inhibits the secretion of growth hormone from the pituitary gland was first reported in 1973 by Roger Guillemin and co-workers, through extraction of the ovine hypothalamus. Later named somatostatin, discovery of this and other hypothalamic hormones resulted in Guillemin being jointly awarded the Nobel Prize for Physiology or Medicine in 1977. Since then, at least 19 studies have been published on the measurement of somatostatin concentration in mammalian blood plasma. Unfortunately, every single reported measurement is wrong, many grossly so, the values ranging from 2.5-360 pg/mL and averaging 60 pg/mL. This current study will detail the measures taken to arrest endogenous somatostatin degradation, selection of a suitable internal standard, relevant technical innovations, and development of LC-MS/MS methods on state-of-the-art instrumentation. Finally, the newly developed analytical method was applied to a pilot study employing ten healthy human subjects. Fasting somatostatin levels were 0.87 ± 0.03 and 0.59 ± 0.11 ng/mL in the male and female cohorts respectively. These concentrations increased to 1.44 ± 0.07 (M) and 1.33 ± 0.18 ng/mL (F) following breakfast

Cu(II) ion directed self-assembly of a Y8/Cu6 heterometallic coordination cage via an Y(III) metalloligand

A non-centrosymmetric yttrium [Y(III)] metalloligand, [Y(H3L)(NO3)]·2NO3·THF (1) {tris{[2-{(imidazole)methylidene}amino]-ethyl}amine = H3L}, was synthesized and subsequently used in a Cu(II) directed self-assembly process to form a Y8/Cu6- type heterometallic coordination cage [Cu6Y8L8(NO3)5(H2O)3]·7NO3·21H2O (2). The eight corners of the distorted cubic cage are defined by eight Y(III) ions while Cu(II) ions occupy the centers of six faces with two opposite Cu(II) ions considerably outside the faces. The asymmetric Y(III) metal centres due to the extra coordination of either water molecules or nitrate anions induce chirality in the cage, with equal numbers of both enantiomers present in the solid state. The cage formation in both solution and in the solid state was demonstrated by ESI-MS and single crystal X-ray diffraction. Magnetic property measurements indicate that cage 2 remains paramagnetic down to 2 K. In addition, vibrational modes, electronic structure and thermal stability of the coordination cage 2 have been further investigated and reported

Development of an inexpensive and non-invasive mass spectrometry based diagnostic test for early stage atherosclerosis

Coronary artery disease (CAD) is the leading cause of death worldwide, accounting for 32% of mortality. In Australia CAD causes 45,000 deaths annually, costing the health system $8 billion per year. CAD is caused by atherosclerosis, the build-up of plaque inside arteries. Diagnosis of CAD presents several problems: it is expensive, imparts a significant radiation load to the patient, and is not sensitive enough to detect the disease in its early stages. We have developed an LC-MS/MS method which looks promising as a sensitive, specific, inexpensive and non-invasive diagnostic test for CAD at a stage where intervention will be effective

Development of a mass spectrometry based diagnostic test for atherosclerosis

Coronary artery disease (CAD) is the leading cause of death worldwide, accounting for 32% of mortality. In Australia CAD causes 45,000 deaths annually, costing the health system $8 billion per year. CAD is caused by atherosclerosis, the build-up of plaque inside arteries. Diagnosis of CAD presents several problems: it is expensive, imparts a significant radiation load to the patient, and is not sensitive enough to detect the disease in its early stages. We are developing an LCMS/MS method which looks promising as a sensitive, specific, inexpensive and non-invasive diagnostic test for CAD at a stage where intervention will be effective

Untargeted proteomic differences between clinical strains of methicillin-sensitive and methicillin-resistant Staphylococcus aureus

Staphylococcus aureus is a common disease-causing bacterium that has developed resistances to a wide variety of antibiotics. This increasing antibiotic resistance has made management of these infections difficult. A better understanding of the general differences among clinical S. aureus strains beyond the well characterized resistance mechanisms may help in identifying new anti-microbial targets. This study aimed to identify and compare the general differences in protein profiles among clinical strains of S. aureus sensitive and resistant to methicillin. The proteomic profiles of five methicillin sensitive (MSSA) and five methicillin resistant (MRSA) S. aureus strains were analyzed by ultra-performance liquid chromatography-mass spectrometry. Protein identification was done using Progenesis QI for Proteomics and the UniProt S. aureus database. Proteins that play roles in virulence, metabolism, and protein synthesis were found to be present at different abundances between MSSA and MRSA (Data available via ProteomeXchange with identifier PXD021629). This study shows differences in protein profiles between antibiotic sensitive and antibiotic resistant clinical strains of S. aureus that may affect the resistance mechanism. Further research on these differences may identify new drug targets against methicillin resistant S. aureus strains

Untargeted lipidomic differences between clinical strains of methicillin-sensitive and methicillin-resistant staphylococcus aureus

Background: Staphylococcus aureus is a common cause of infectious diseases in humans. It has become resistant to many antibacterial agents making management of infections difficult. A better understanding of differences among S. aureus strains that are sensitive and resistant to antibiotics may offer insights into the resistant phenotype and identify new antimicrobial targets. This study aimed at comparing general differences in lipid profiles among clinical strains of S. aureus sensitive and resistant to antibiotics. The cell wall thickness and cell surface charge were also compared. Methods: Five methicillin sensitive (MSSA) and five methicillin resistant (MRSA) S. aureus strains were compared both individually and as MSSA and MRSA groups in the absence of antibiotics. Lipids were compared by ultra-performance liquid chromatography-mass spectrometry, cell wall thickness was compared by scanning transmission electron microscopy and whole-cell surface charge was compared using a cytochrome c binding assay. Results: Twenty-two lipid species were identified in all ten strains of S. aureus. The abundance of three lipid species (two lysyl-phosphatidylglycerol and one diglycosyldiacylglycerol) were found to be different between MSSA and MRSA. Differences in cell wall thickness were identified between strains but not between MSSA and MRSA. No difference in whole-cell surface charge was observed between MSSA and MRSA. Conclusion: This study shows differences in membrane lipids between antibiotic sensitive and antibiotic resistant clinical strains of S aureus that may affect resistance mechanisms related to cell membrane structure and fluidity. Further research on these differences may identify new drug targets against resistant strains

Journal of abnormal psychology

Poly(3,4-ethylenedioxythiophene) (PEDOT) is an organic conducting polymer that has been the focus of significant research over the last decade, in both energy and biological applications. Most commonly, PEDOT is doped by the artificial polymer polystyrene sulfonate due to the excellent electrical characteristics yielded by this pairing. The biopolymer dextran sulphate (DS) has been recently reported as a promising alternative to PEDOT:PSS for biological application, having electrical properties rivaling PEDOT:PSS, complimented by the potential bioactivity of the polysaccharide. In this work we compared chemical and electrochemical polymerisations of PEDOT:DS in terms of their impact on the electrical, morphological and biological properties of the resultant PEDOT:DS films. Post-growth cyclic voltammograms and UV-Vis analyses revealed comparable redox behaviour and absorbance profiles for the two synthesis approaches. Despite good intrinsic conductivity of particles, the addition of chemically produced PEDOT:DS did not markedly enhance the bulk conductivity of aqueous solutions due to the lack of interconnectivity between adjacent PEDOT:DS particles at achievable concentrations. Scanning electron microscopy revealed significantly greater roughness in films cast from chemically produced PEDOT:DS compared to electropolymerised samples, attributable to the formation of solution phase nanoparticles prior to casting. In cell studies with the L929 cell line, electrochemical polymerisation of PEDOT:DS afforded better integrity of resultant films for surface seeding, whilst chemically polymerised PEDOT:DS appeared to localised at the proliferating cells, suggesting possible applications in drug delivery