Introduction: Increasing evidence suggests a role for the gut microbiome in central nervous system disorders and specific role for the gut-brain axis in neurodegeneration. Bile acids (BA), products of cholesterol metabolism and clearance, are produced in the liver and are further metabolized by gut bacteria. They have major regulatory and signaling functions and seem dysregulated in Alzheimer disease (AD). Methods: Serum levels of 15 primary and secondary BAs and their conjugated forms were measured in 1,464 subjects including 370 cognitively normal older adults (CN), 284 with early mild cognitive impairment (MCI), 505 with late MCI, and 305 AD cases enrolled in the AD Neuroimaging Initiative. We assessed associations of BA profiles including selected ratios with diagnosis, cognition, and AD-related genetic variants, adjusting for cofounders and multiple testing. Results: In AD compared to CN, we observed significantly lower serum concentrations of a primary BA (cholic acid CA) and increased levels of the bacterially produced, secondary BA, deoxycholic acid (DCA), and its glycine and taurine conjugated forms. An increased ratio of DCA:CA, which reflects 7α-dehydroxylation of CA by gut bacteria, strongly associated with cognitive decline, a finding replicated in serum and brain samples in the Rush Religious Orders and Memory and Aging Project. Several genetic variants in immune response related genes implicated in AD showed associations with BA profiles. Conclusion: We report for the first time an association between altered BA profile, genetic variants implicated in AD and cognitive changes in disease using a large multicenter study. These findings warrant further investigation of gut dysbiosis and possible role of gut liver brain axis in the pathogenesis of AD.

The influx of genomic sequence information has led to the concept of structural proteomics, the determination of protein structures on a genome-wide scale. Here we describe an approach to structural proteomics of small proteins using NMR spectroscopy. Over 500 small proteins from several organisms were cloned, expressed, purified, and evaluated by NMR. Although there was variability among proteomes, overall 20% of these proteins were found to be readily amenable to NMR structure determination. NMR sample preparation was centralized in one facility, and a distributive approach was used for NMR data collection and analysis. Twelve structures are reported here as part of this approach, which allowed us to infer putative functions for several conserved hypothetical proteins. S tructural proteomics, which aims to determine the threedimensional (3D) structures of all proteins, has become a major initiative within the biomedical community (see ref. 1 and other articles in the same issue). The large number of protein structures expected from these projects will yield valuable clues to the rules for predicting protein folding and understanding biochemical function. In these early stages of the structural proteomics effort, one of the main goals is to identify the best technologies and the most efficient processes to convert gene sequence into 3D structural information. One of the decisions will be to determine the optimal use of x-ray crystallography and NMR spectroscopy, which are the two techniques that will provide the majority of experimental data for these initiatives.X-ray crystallography currently is perceived as the potential workhorse for structural proteomics, because if provided with a well diffracting crystal it is possible to determine a 3D structure in hours. However, the throughput of structure determination using x-ray crystallography remains unclear, because the ratedetermining step continues to be the production of well diffracting crystals, a process that is unpredictable and can take between hours and months.NMR structure determination is limited currently by size constraints and lengthy data collection and analysis times (often months), and the method is best applied to proteins smaller than 250 amino acids. On the other hand, NMR experiments do not require crystals, and samples appropriate for structure determination can be identified within minutes of the protein being purified. In summary, x-ray crystallography and NMR spectroscopy seem to have complementary deficiencies, and the relative success of these methods in structural proteomics remains to be determined.We have shown previously that NMR spectroscopy can play a significant role in structural proteomics even with its current limitations (2). The initial pilot project, based on a limited number of proteins from the thermophilic archaebacterium Methanobacterium thermoautotrophicum (Mth) suggested that smaller proteins may be more amenable to structure analysis, because in this genome a higher proportion of smaller proteins were soluble compar...

1 A 12 week prospective survey of all patients of any age with confirmed or suspected poisoning presenting to six accident and emergency departments in the North East of England was performed to establish the local incidence and patterns of presentation of poisoning. 2 945 episodes of poisoning involving 852 patients were recorded representing approximately 1.2% of all A&E presentations and suggesting an annual attendance rate of 2.7 per 1000 persons per year. 3 Attendance rates varied threefold between hospitals and were similar in males and females overall; between the ages of five and 14 attendances were more common in females (1.9 vs 0.6/103/y) while between 0 and 4 y (3.1 vs 2.4) and 25 and 34 y (3.9 vs 2.9/103/y) they were more common in males. 4 The median interval between poisoning and presenta tion was 2 h (mean 4.1 h) and only 19% of cases presented within 1 h. Presentation was most common between Friday evening and Tuesday morning and in the late afternoon and evening. 5 6% of the patients presented more than once with poisoning during the study period and 37% had a past history of deliberate self-harm. The most common poisons involved were paracetamol (43%), opioids (15%) and benzodiazepines (15%). 6 The study illustrates the frequency of presentations of poisoning to A&E departments. The high rate of poisoning in young men and the increasing use of paracetamol are particular causes for concern.

In the United States, mortality rates from pancreatic cancer (PCa) have not changed significantly over the past 50 years. This is due, in part, to the lack of early detection methods for this particularly aggressive form of cancer. The objective of this study was to use high-throughput protein profiling technology to identify biomarkers in the serum proteome for the early detection of resectable PCa. Using surface-enhanced laser desorption/ionization mass spectrometry, protein profiles were generated from sera of 49 PCa patients and 54 unaffected individuals after fractionation on an anion exchange resin. The samples were randomly divided into a training set (69 samples) and test set (34 samples), and two multivariate analysis procedures, classification and regression tree and logistic regression, were used to develop classification models from these spectral data that could distinguish PCa from control serum samples. In the test set, both models correctly classified all of the PCa patient serum samples (100% sensitivity). Using the decision tree algorithm, a specificity of 93.5% was obtained, whereas the logistic regression model produced a specificity of 100%. These results suggest that high-throughput proteomics profiling has the capacity to provide new biomarkers for the early detection and diagnosis of PCa.

Developing biomarkers for detecting acetaminophen (APAP) toxicity has been widely investigated. Recent studies of adults with APAP-induced liver injury have reported human serum microRNA-122 (miR-122) as a novel biomarker of APAP-induced liver injury. The goal of this study was to examine extracellular microRNAs (miRNAs) as potential biomarkers for APAP liver injury in children. Global levels of serum and urine miRNAs were examined in three pediatric subgroups: 1) healthy children (n=10), 2) hospitalized children receiving therapeutic doses of APAP (n=10) and 3) children hospitalized for APAP overdose (n=8). Out of 147 miRNAs detected in the APAP overdose group, eight showed significantly increased median levels in serum (miR-122, −375, −423-5p, −30d-5p, −125b-5p, −4732-5p, −204-5p, and −574-3p), compared to the other groups. Analysis of urine samples from the same patients had significantly increased median levels of four miRNAs (miR-375, −940, −9-3p and −302a) compared to the other groups. Importantly, correlation of peak serum APAP protein adduct levels (an indicator of the oxidation of APAP to the reactive metabolite N-acetylpara-quinone imine) with peak miRNA levels showed that the highest correlation was observed for serum miR-122 (R=0.94; p<0.01) followed by miR-375 (R=0.70; p=0.05). Conclusion: Our findings demonstrate that miRNAs are increased in children with APAP toxicity and correlate with APAP protein adducts, suggesting a potential role as biomarkers of APAP toxicity.