Proteomic profiling of neuromas reveals alterations in protein composition and local protein synthesis in hyper-excitable nerves

Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis ( 2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a > 1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel alpha 2 delta-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S(35)methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability

A targeted glycoproteomic approach identifies cadherin-5 as a novel biomarker of metastatic breast cancer

Aberrant glycosylation has long been recognised as a hallmark of cancer, and is increasingly being exploited in biomarker discovery studies. Helix pomatia agglutinin (HPA) is known to bind aberrant glycans associated with metastatic breast cancer, and was used here to isolate glycoproteins from pooled breast cancer serum samples of (i) patients with recurrent breast cancer and (ii) patients with no sign of recurrence 5 years after diagnosis of their primary tumour. Pregnancy zone protein, the polymeric immunoglobulin receptor and cadherin-5 emerged as potential markers of metastasis following proteomic identification of HPA binding glycoproteins. ELISAs were developed to verify these findings, and to assess protein glycosylation, in individual patient sera. The cadherin-5 ELISA discriminated serum samples of patients with recurrent breast cancer from those with no sign of recurrence, and analysis of cadherin-5 glycosylation by HPA also showed a significant difference between the two sample groups. The targeted glycoproteomic and validatory approach developed here has shown that when taking into account both the protein levels and HPA binding, serum cadherin-5 discriminated patients with recurrent breast cancer from those with no sign of recurrence with 90% specificity

Ages of lunar impact breccias: Limits for timing of the Imbrium impact

Since the Apollo 14 mission delivered samples of the Fra Mauro formation, interpreted as ejecta of the Imbrium impact, defining the age of this impact has emerged as one of the critical tasks required for the complete understanding of the asteroid bombardment history of the Moon and, by extension, the inner Solar System. Significant effort dedicated to this task has resulted in a substantial set of ages centered around 3.9 Ga and obtained for the samples from most Apollo landing sites using a variety of chronological methods. However, the available age data are scattered over a range of a few tens of millions of years, which hinders the ability to distinguish between the samples that are truly representative of the Imbrium impact and those formed/reset by other, broadly contemporaneous impact events. This study presents a new set of U-Pb ages obtained for the VHK (very high K) basalt clasts found in the Apollo 14 breccia sample 14305 and phosphates from (i) several fragments of impact-melt breccia extracted from Apollo 14 soil sample 14161, and (ii) two Apollo 15 breccias 15455 and 15445. The new data obtained for the Apollo 14 samples increase the number of independently dated samples from this landing site to ten. These Apollo 14 samples represent the Fra Mauro formation, which is traditionally viewed as Imbrium ejecta, and therefore should record the age of the Imbrium impact. Using the variance of ten ages, we propose an age of 3922 ± 12 Ma for this event. Samples that yield ages within these limits can be considered as possible products of the Imbrium impact, while those that fall significantly outside this range should be treated as representing different impact events. Comparison of this age for Imbrium (determined from Apollo 14 samples) with the ages of another eleven impact-melt breccia samples collected at four other landing sites and a related lunar meteorite suggests that they can be viewed as part of Imbrium ejecta. Comprehensive review of 40Ar/39Ar ages available for impact melt samples from different landing sites and obtained using the step-heating technique, suggests that the majority of the samples that gave robust plateau ages are indistinguishable within uncertainties and altogether yield a weighted average age of 3916 ± 7 Ma (95 % conf., MSWD = 1.1; P = 0.13) and a median average age of 3919 + 14/-12 Ma, both of which agree with the confidence interval obtained using the U-Pb system. These samples, dated by 40Ar/39Ar method, can be also viewed as representing the Imbrium impact. In total 36 out of 41 breccia samples from five landing sites can be interpreted to represent formation of the Imbrium basin, supporting the conclusion that Imbrium material was distributed widely across the near side of the Moon. Establishing temporal limits for the Imbrium impact allows discrimination of ten samples with Rb-Sr and 40Ar/39Ar ages about 50 Ma younger than 3922 ± 12 Ma. This group may represent a separate single impact on the Moon and needs to be investigated further to improve our understanding of lunar impact history