The secreted phospholipase A2 human group IIA (hGIIA) is overexpressed in prostate cancer (PCa), where its expression is closely aligned with malignancy. While its enzymatic activity is important in mediating innate immunity, here we highlight that hGIIA contributes to PCa pathology primarily through specific protein-protein interactions. We have developed cyclic peptides cF and c2, derived from the structure of hGIIA, that selectively inhibit these interactions and inhibit PCa growth. hGIIA interacts directly with epidermal growth factor receptor (EGFR), resulting in increased cytosolic PLA2-α activation and prostaglandin E2 production, which is suppressed by c2. Further, vimentin was identified to bind hGIIA in PCa cells, modulating hGIIA intracellular trafficking. c2 binds vimentin, blocking this interaction and initiating vimentin-mediated aggresome formation and apoptosis even in the absence of hGIIA. cF and c2 suppress androgen-sensitive, castrate-resistant and androgen-independent models of tumour growth in vivo at doses as low as 0.1 mg/kg, are non-toxic, orally bioavailable and cell-permeable. Critically, as with hGIIA, EGFR and vimentin are also increasingly expressed as PCa develops, cF and c2 may represent a novel therapeutic option for incurable metastatic castrate resistant PCa. Our findings identify hGIIA as an innate immune effector that regulates both inflammation and PCa progression and describe a novel class of hGIIA protein-protein interaction inhibitor with therapeutic potential in PCa.

Although electrostatic catalysis can enhance the kinetics and selectivity of reactions to produce greener synthetic processes, the highly directional nature of electrostatic interactions has limited widespread application. In this study, the influence of oriented electric fields (OEF) on radical addition and atom abstraction reactions are systematically explored with ion-trap mass spectrometry using structurally diverse distonic radical ions that maintain spatially separated charge and radical moieties. When installed on rigid molecular scaffolds, charged functional groups lock the magnitude and orientation of the internal electric field with respect to the radical site, creating an OEF which tunes the reactivity across the set of gas-phase carbon-centred radical reactions. In the first case, OEFs predictably accelerate and decelerate the rate of molecular oxygen addition to substituted phenyl, adamantyl, and cubyl radicals, depending on the polarity of the charged functional group and dipole orientation. In the second case, OEFs modulate competition between chlorine and hydrogen atom abstraction from chloroform based on interactions between charge polarity, dipole orientation, and radical polarizability. Importantly, this means the same charge polarity can induce different changes to reaction selectivity. Quantum chemical calculations of these reactions with DSD-PBEP86-D3(BJ)/aug-cc-pVTZ show correlations between the barrier heights and the experimentally determined reaction kinetics. Field effects are consistent between phenyl and cubyl scaffolds, pointing to through-space rather than through-bond field effects, congruent with computations showing that the same effects can be mimicked by point charges. These results experimentally demonstrate how internal OEFs generated by carefully placed charged functional groups can systematically control radical reactions.

Anti-inflammatory bioassay-guided compound isolation from the exocarp of the Australian rainforest tree Endiandra insignis (family Lauraceae) has led to the discovery and structural elucidation of unusual alpha, beta-unsaturated twenty-four carbon fatty acids and their positional isomers, insignoic acids A - E (1a - 5c). The stereochemistry and position of the double bond within the aliphatic chain were independently determined via NMR spectroscopy and Ozone-Induced Dissociation (OzID) Mass Spectrometry, respectively. Compounds (1a - 5c) displayed good to moderate anti-inflammatory activity in the range of 8-84 mu M. The low therapeutic index observed when assessing the cell viability in the RAW macrophage cell lines, prompted us to investigate the anticancer potential of these unusual fatty acids. The anti-cancer activity was assessed in A-431 carinoma cell lines and MM649 melanoma cell lines. Insignoic acid C (3a-f) exhibited the highest level of potency with an IC50 value of 5-7 mu M against both the cell lines. The insignoic acids are the first of their kind known for incorporating an alpha-beta unsaturated system flanked next to a keto group with an additional level of oxygenation at C-6 in a 24-carbon fatty acid backbone.

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.

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.

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 binding interactions of a series of square-planar platinum(II)–phenanthroline complexes of the type [Pt(PL)(AL)]2+ [where PL = variously methyl-substituted 1,10-phenanthroline (phen) and AL = ethane-1,2-diamine (en)] were assessed with a G-quadruplex DNA (5′-TTG GGG GT-3′, G4DNA) and a double-stranded DNA (5′-CGC GAA TTC GCG-3′, dsDNA) sequence by ESI–MS. The results indicate a strong correlation between G4DNA affinity and increasing phenanthroline methyl substitution. Circular dichroism (CD) spectroscopy and molecular docking studies also support the finding that increased substitution of the phenanthroline ligand increased selectivity for G4DNA. ESI-MS was used to probe the interaction of a range of square-planar Pt(II)–phenanthroline complexes with double-stranded and G-quadruplex DNA.

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 producerDoreaare 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, andStreptococcus), 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.

Through a screen of over one hundred and 30 permutations of reaction temperatures, solvents, carbodiimide resins, and carbodiimide molar equivalences, in the presence, absence, or combination of diisopropylamine and benzotriazole additives, a convenient and first reported carbodiimide polymer-assisted flow approach to effect amide coupling and lactamization was developed. The protocol entails injecting a single solution (1:9 dimethylformamide: dichloromethane) containing a carboxylic acid and an amine or linear peptide sequence into a continuous stream of dichloromethane. The protocol remained viable in the absence of base, did not require carboxylate preactivation which, and in concert with minimal workup requirements, enabled the isolation of products in high yields. Compared to the utilization of untethered carbodiimide reagents, the flow procedure was also observed to provide a degree of racemization safety.

Here we describe a modified method for harvesting tens-of-millions of human lens epithelial-like cells from differentiated pluripotent stem cell cultures. To assess the utility of this method, we analysed the lens cell population via: light microscopy; single cell RNA-sequencing and gene ontology analyses; formation of light-focusing micro-lenses; mass spectrometry; and electron microscopy. Both individually and collectively, the data indicate this simplified harvesting method provides a large-scale source of stem cell-derived lens cells and micro-lenses for investigating human lens and cataract formation.

Glyoxal (GO) and methylglyoxal (MGO) are two important biomarkers in diabetes. Analytical methods for determination of GO and MGO in serum samples are either HPLC with UV-Vis (low sensitivity) or MS/MS (expensive) detection. These disadvantages have hampered the introduction of these biomarkers as a routine analyte for diabetes diagnostics into the clinical laboratory. In this study, we introduce a UHPLC method with fluorescence detection for the measurement of GO and MGO in serum samples by pre-column derivatization at neutral pH with 5, 6-diamino-2,4-dihydroxypyrimidine sulfate (DDP) to form lumazines. The method was validated as per FDA guidelines. Using this method, we have determined GO and MGO in a variety of animal serum samples, and for example, determined the GO and MGO concentration in adult bovine serum to be 852 +/- 27 and 192 +/- 10 nmol/L, respectively. In human serum, GO and MGO levels in non-diabetic subjects (n = 14) were determined to be 154 +/- 88 and 98 +/- 27 nmol/L, and in serum samples from subjects with diabetes (n = 14) 244 +/- 137 and 190 +/- 68 nmol/L, respectively. In addition, interference studies showed that physiological serum components did not lead to an artificial increase in the levels of GO and MGO.

A non-centrosymmetric yttrium [Y(III)] metalloligand, [Y(H3L)(NO3)]center dot 2NO(3)center dot THF (1) {tris{[2-{(imidazole)methylidene}aminol-ethyllamine = 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)]center dot 7NO(3)center dot 21H(2)O (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.

The pathogenesis of synucleinopathies, common neuropathological lesions normally associated with some human neurodegenerative disorders such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, remains poorly understood. In animals, ingestion of the tryptamine-alkaloid-rich phalaris pastures plants causes a disorder called Phalaris staggers, a neurological syndrome reported in kangaroos. The aim of the study was to characterise the clinical and neuropathological changes associated with spontaneous cases of Phalaris staggers in kangaroos. Gross, histological, ultrastructural and Immunohistochemical studies were performed to demonstrate neuronal accumulation of neuromelanin and aggregated α-synuclein. ELISA and mass spectrometry were used to detect serum-borne α-synuclein and tryptamine alkaloids respectively. We report that neurons in the central and enteric nervous systems of affected kangaroos display extensive accumulation of neuromelanin in the perikaryon without affecting neuronal morphology. Ultrastructural studies confirmed the typical structure of neuromelanin. While we demonstrated strong staining of α-synuclein, restricted to neurons, intracytoplasmic Lewy bodies inclusions were not observed. α-synuclein aggregates levels were shown to be lower in sera of the affected kangaroos compared to unaffected herd mate kangaroos. Finally, mass spectrometry failed to detect the alkaloid toxins in the sera derived from the affected kangaroos. Our preliminary findings warrant further investigation of Phalaris staggers in kangaroos, potentially a valuable large animal model for environmentally-acquired toxic synucleinopathy.

From a screen of chlorinating agent concentration, flow rates, and reagent addition methodologies, a continuous flow protocol to activate, reactivate, and recycle both 2-chlorotrityl chloride functionalised polystyrene and trityl-hydroxy ChemMatrix functionalised resins was established.

Cataracts cause vision loss and blindness by impairing the ability of the ocular lens to focus light onto the retina. Various cataract risk factors have been identified, including drug treatments, age, smoking and diabetes. However, the molecular events responsible for these different forms of cataract are ill-defined, and the advent of modern cataract surgery in the 1960s virtually eliminated access to human lenses for research. Here, we demonstrate large-scale production of light-focusing human micro-lenses from spheroidal masses of human lens epithelial cells purified from differentiating pluripotent stem cells. The purified lens cells and micro-lenses display similar morphology, cellular arrangement, mRNA expression and protein expression to human lens cells and lenses. Exposing the microlenses to the emergent cystic fibrosis drug Vx-770 reduces micro-lens transparency and focusing ability. These human micro-lenses provide a powerful and large-scale platform for defining molecular disease mechanisms caused by cataract risk factors, for anti-cataract drug screening and for clinically relevant toxicity assays.

An Intramolecular Diels-Alder cycloaddition reaction between a furan diene and an alkynic dienophile was performed within a flow hydrogenator fitted with an inert titanium column at 150 degrees C, no H-2, under 100bar pressure. A single column pass (t(R)=1.6min) afforded approximate to 55% conversion to the tetrahydroepoxyisoindole carboxamide scaffold with a product turnover of approximate to 0.035g/h, a 95% improvement over batch procedures. The cycloaddition protocol is performed in water and ethanol, and does not require catalysts or other additives. Quantitative hydrogenation of the resulting dual -bonds within the oxabicyclo system was effected with either a 10% Pd/Al2O3 or 10% Pd/CaCO3 catalyst at 20 degrees C, 20bar, with full H-2 whereas utilisation of a Raney Ni catalyst under these conditions resulted in a quantitative mono-olefin reduction of the C4-C5 double-bond. With regard to di-olefin reduction, a degree of stereoselectivity was observed with the aforementioned palladium-based catalysts yielding exclusive formation of (R) configuration at C7 while utilisation of a 5% Pt/C (sulfided) catalyst at temperatures below 60 degrees C promoted the formation of the (S)-isomer at C7. Hence this work further highlights that flow-hydrogenation provides unprecedented convenience for establishing robust protocols to effect chemoselective transformations.

From the repurposing of used HPLC components, a chromatography column, a Rheodyne,® and a heater stirrer, a simple, cost-effective system to conveniently conduct continuous flow solid-phase peptide construction was assembled.

Measurement of dopamine (DA) release in the retina allows the interrogation of the complex neural circuits within this tissue. A number of previous methods have been used to quantify this neuromodulator, the most common of which is HPLC with electrochemical detection (HPLC-ECD). However, this technique can produce significant concentration uncertainties. In this present study, we report a sensitive and accurate UHPLC-MS/MS method for the quantification of DA and its primary metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in mouse retina. Internal standards DA-d(4) and DOPAC-d(5) result in standard curve linearity for DA from 0.05-100 ng/mL (LOD = 6 pg/mL) and DOPAC from 0.5-100 ng/mL (LOD = 162 pg/mL). A systematic study of tissue extraction conditions reveals that the use of formic acid (1%), in place of the more commonly used perchloric acid, combined with 0.5 mM ascorbic acid prevents significant oxidation of the analytes. When the method is applied to mouse retinae a significant increase in the DOPAC/DA ratio is observed following in vivo light stimulation. We additionally examined the effect of anesthesia on DA and DOPAC levels in the retina in-vivo and find that basal dark-adapted concentrations are not affected. Light caused a similar increase in DOPAC/DA ratio but interindividual variation was significantly reduced. Together, we systematically describe the ideal conditions to accurately and reliably measure DA turnover in the mammalian retina.

A series of 28 norcantharidin (NorC)-inspired analogues were accessed via a robust two-step Ugi intramolecular Diels-Alder (IMDA) sequence. Four analogues displayed whole-cell cytotoxicity equipotent to that of NorC and cisplatin against a number of cancer cell lines and a normal breast cell line (MCF10A). Notably, (3S,3aS,6R)-2-benzyl-7-methyl-N-(naphthalen-2-yl)-1-oxo-1,2,3,6-tetrahydro-3a,6-epoxyisoindole-3-carboxamide (trans-27) displayed superior whole-cell activity against breast (MCF-7, GI50 =2.9 μm) and colon (HT29, GI50 =6.4 μm) cancer cell lines relative to the control (cisplatin), which elicited respective GI50 values of 6.5 and 11.3 μm against the aforementioned cell lines. This analogue also displayed improved activity relative to NorC across the breast (MCF-7, GI50 =2.9 μm; NorC GI50 =7.5 μm), ovarian (A2780, GI50 =2.2 μm; NorC GI50 =4.4 μm), and neuroblastoma (BE2-C, GI50 =2.2 μm; NorC GI50 =3.7 μm) cancer cell lines. Structure-activity relationship (SAR) investigations demonstrated that retention of sp2 hybridized connections within the tetrahydroepoxyisoindole carboxamide scaffold is crucial, as aromatization to a phenolic functionality decreased activity, whereas removal of a single olefin bond abolished cytotoxicity. Nonetheless, with respect to the latter, use of crotonic acid as opposed 2-butynoic acid in the Ugi-IMDA sequence imparted a significant improvement to diastereoselectivity, with the cis/trans isomer ratio shifting from ≈1:1.2 to ≈0.5:9.5.

We present the synthesis and characterization of a unique, slightly distorted square prismatic, box-like coordination cage of type [Cu6Dy8L8(MeOH)8(H2O)6](NO3)12·χsolvent obtained via the supramolecular assembly between a non-centrosymmetric Dy(iii) metalloligand and Cu(ii) nitrate. Magnetic susceptibility measurements indicate that the complex behaves as a single-molecule magnet.