Titrations without the Additions: The Efficient Determination of pKa Values Using NMR Imaging Techniques

It can be very informative to acquire NMR spectra of a sample as a function of the solution pH. Examples can be found in the design of host–guest complexes or in the determination of the pKa values of organic molecules. In the conventional procedure, a series of spectra must be recorded and the pH of the sample adjusted manually between successive NMR measurements. As an alternative to this laborious procedure, we demonstrate how controlled pH gradients may be established in 5 mm NMR tubes and analyzed using standard NMR equipment in a “single shot” experiment. Using 1H NMR imaging techniques and a set of NMR pH indicator compounds, we are able to measure the pH of a sample as a function of position along a pH gradient. We are thus able to obtain the necessary set of 1H NMR spectra as a function of pH from a single sample in a single NMR experiment. As proof of concept, we demonstrate how the technique may be employed for the determination of the pKa values of small organic molecules. We are able to measure pKa values from 1 to 11 to within 0.1 units of their literature values. The method is robust to variations in the setting of the pH gradients and can be readily implemented through an automated sample changer

HAGE (DDX43) is a biomarker for poor prognosis and a predictor of chemotherapy response in breast cancer

Background: HAGE protein is a known immunogenic cancer-specific antigen. Methods: The biological, prognostic and predictive values of HAGE expression was studied using immunohistochemistry in three cohorts of patients with BC (n=2147): early primary (EP-BC; n=1676); primary oestrogen receptor-negative (PER-BC; n=275) treated with adjuvant anthracycline-combination therapies (Adjuvant-ACT); and primary locally advanced disease (PLA-BC) who received neo-adjuvant anthracycline-combination therapies (Neo-adjuvant-ACT; n=196). The relationship between HAGE expression and the tumour-infiltrating lymphocytes (TILs) in matched prechemotherapy and postchemotherapy samples were investigated. Results: Eight percent of patients with EP-BC exhibited high HAGE expression (HAGEþ) and was associated with aggressive clinico-pathological features (Ps<0.01). Furthermore, HAGEþexpression was associated with poor prognosis in both univariate and multivariate analysis (Ps<0.001). Patients with HAGE+ did not benefit from hormonal therapy in high-risk ER-positive disease. HAGE+ and TILs were found to be independent predictors for pathological complete response to neoadjuvant-ACT; P<0.001. A statistically significant loss of HAGE expression following neoadjuvant-ACT was found (P=0.000001), and progression-free survival was worse in those patients who had HAGE+ residual disease (P=0.0003). Conclusions: This is the first report to show HAGE to be a potential prognostic marker and a predictor of response to ACT in patients with BC

Clinical and genomic analysis of a randomised phase II study evaluating anastrozole and fulvestrant in postmenopausal patients treated for large operable or locally-advanced hormone-receptor-positive breast cancer

Background: The aim of this study was to assess the efficacy of neoadjuvant anastrozole and fulvestrant treatment of large operable or locally-advanced hormone- receptor-positive breast cancer not eligible for initial breast-conserving surgery, and to identify genomic changes occurring after treatment. Methods: 120 post-menopausal patients were randomised to receive 1 mg anastrozole (61 patients) or 500 mg fulvestrant (59 patients) for 6 months. Genomic DNA copy number profiles were generated for a subgroup of 20 patients before and after treatment. Results: 108 patients were evaluable for efficacy and 118 for toxicity. The objective response rate determined by clinical palpation was 58.9% (95% CI 45.0-71.9) in the anastrozole arm and 53.8% (95% CI 39.5-67.8) in the fulvestrant arm. The breast- conserving surgery rate was 58.9% (95% CI 45.0-71.9) in the anastrozole arm and 50.0% (95% CI 35.8-64.2) in the fulvestrant arm. Pathological responses >50% occurred in 24 patients (42.9%) in the anastrozole arm and 13 (25.0%) in the fulvestrant arm. The Ki-67 score fell after treatment but there was no significant difference between the reduction in the two arms (anastrozole 16.7% [95%CI 13.3-21.0] before, 3.2% [95%CI 1.9-5.5] after, n=43; fulvestrant 17.1% [95%CI 13.1-22.5] before, 3.2% [95%CI 1.8-5.7] after, n=38) or between the reduction in Ki-67 in clinical responders and non- responders. Genomic analysis appeared to show a reduction of clonal diversity following treatment with selection of some clones with simpler copy number profiles. Conclusion: Both anastrozole and fulvestrant were effective and well-tolerated, enabling breast-conserving surgery in over 50% of patients. Clonal changes consistent with clonal selection by the treatment were seen in a subgroup of patients

Psychophysical Investigations into the Role of Low-Threshold C Fibres in Non-Painful Affective Processing and Pain Modulation

We recently showed that C low-threshold mechanoreceptors (CLTMRs) contribute to touch-evoked pain (allodynia) during experimental muscle pain. Conversely, in absence of ongoing pain, the activation of CLTMRs has been shown to correlate with a diffuse sensation of pleasant touch. In this study, we evaluated (1) the primary afferent fibre types contributing to positive (pleasant) and negative (unpleasant) affective touch and (2) the effects of tactile stimuli on tonic muscle pain by varying affective attributes and frequency parameters. Psychophysical observations were made in 10 healthy participants. Two types of test stimuli were applied: stroking stimulus using velvet or sandpaper at speeds of 0.1, 1.0 and 10.0 cm/s; focal vibrotactile stimulus at low (20 Hz) or high (200 Hz) frequency. These stimuli were applied in the normal condition (i.e. no experimental pain) and following the induction of muscle pain by infusing hypertonic saline (5%) into the tibialis anterior muscle. These observations were repeated following the conduction block of myelinated fibres by compression of sciatic nerve. In absence of muscle pain, all participants reliably linked velvet-stroking to pleasantness and sandpaper-stroking to unpleasantness (no pain). Likewise, low-frequency vibration was linked to pleasantness and high-frequency vibration to unpleasantness. During muscle pain, the application of previously pleasant stimuli resulted in overall pain relief, whereas the application of previously unpleasant stimuli resulted in overall pain intensification. These effects were significant, reproducible and persisted following the blockade of myelinated fibres. Taken together, these findings suggest the role of low-threshold C fibres in affective and pain processing. Furthermore, these observations suggest that temporal coding need not be limited to discriminative aspects of tactile processing, but may contribute to affective attributes, which in turn predispose individual responses towards excitatory or inhibitory modulation of pain

Polyanionic Ligand Platforms for Methyl- and Dimethylaluminum Arrays

Trimethylaluminum finds widespread applications in chemical and materials synthesis, most prominently in its partially hydrolyzed form of methylalumoxane (MAO), which is used as a cocatalyst in the polymerization of olefins. This work investigates the sequential reactions of trimethylaluminum with hexaprotic phosphazenes (RNH)6P3N3 (=XH6) equipped with substituents R of varied steric bulk including tert-butyl (1H6), cyclohexyl (2H6), isopropyl (3H6), isobutyl (4H6), ethyl (5H6), propyl (6H6), methyl (7H6), and benzyl (8H6). Similar to MAO, the resulting complexes of polyanionic phosphazenates [XHn]n−6 accommodate multinuclear arrays of [AlMe2]+ and [AlMe]2+. Reactions were monitored by 31P NMR spectroscopy, and structures were determined by single-crystal X-ray diffraction. They included 1H4(AlMe2)2, 1H3(AlMe2)3, 2H3(AlMe2)3, 3(AlMe2)4AlMe, 4H­(AlMe2)5, 4(AlMe2)6, {5H­(AlMe2)4}2AlMe, 5(AlMe2)6, 6(AlMe2)6, {7(AlMe2)4AlMe}2, and 8(AlMe2)6. The study shows that subtle variations of the steric properties of the R groups influence the reaction pathways, levels of aggregation, and fluxional behavior. While [AlMe2]+ is the primary product of the metalation, [AlMe]2+ is utilized to alleviate overcrowding or to aid aggregation. At the later stages of metalation, [AlMe2]+ groups start to scramble around congested sites. The ligands proved to be very robust and extremely flexible, offering a unique platform to study complex multinuclear metal arrangements

Extrinsic primary afferent signalling in the gut

Visceral sensory neurons activate reflex pathways that control gut function and also give rise to important sensations, such as fullness, bloating, nausea, discomfort, urgency and pain. Sensory neurons are organised into three distinct anatomical pathways to the central nervous system (vagal, thoracolumbar and lumbosacral). Although remarkable progress has been made in characterizing the roles of many ion channels, receptors and second messengers in visceral sensory neurons, the basic aim of understanding how many classes there are, and how they differ, has proven difficult to achieve. We suggest that just five structurally distinct types of sensory endings are present in the gut wall that account for essentially all of the primary afferent neurons in the three pathways. Each of these five major structural types of endings seems to show distinctive combinations of physiological responses. These types are: 'intraganglionic laminar' endings in myenteric ganglia; 'mucosal' endings located in the subepithelial layer; 'muscular–mucosal' afferents, with mechanosensitive endings close to the muscularis mucosae; 'intramuscular' endings, with endings within the smooth muscle layers; and 'vascular' afferents, with sensitive endings primarily on blood vessels. 'Silent' afferents might be a subset of inexcitable 'vascular' afferents, which can be switched on by inflammatory mediators. Extrinsic sensory neurons comprise an attractive focus for targeted therapeutic intervention in a range of gastrointestinal disorders.Australian National Health and Medical Research Counci

Efficient pKa Determination in a Nonaqueous Solvent Using Chemical Shift Imaging

Efficient pKa Determination in a Nonaqueous Solvent Using Chemical Shift Imagin

Modular Synthesis of Bicyclic Twisted Amides and Anilines

Bridged amides and anilines display interesting properties owing to perturbation of conjugation of the nitrogen lone-pair with the adjacent π-system. A convergent approach to diazabicyclic scaffolds which contain either twisted amides or anilines is described, based on the photocatalysed hydroamination of cyclic enecarbamates and subsequent cyclisation. The modular nature of the synthesis allows for variation of the degree of ‘twist’ and hence the properties of the amides and anilines

Methanol as hydrogen source: transfer hydrogenation of aromatic aldehydes with a rhodacycle

A cyclometalated rhodium complex has been shown to perform highly selective and efficient reduction of aldehydes, deriving the hydrogen from methanol. With methanol as both the solvent and hydrogen donor under mild conditions and an open atmosphere, a wide range of aromatic aldehydes were reduced to the corresponding alcohols, without affecting other functional groups