Urine-based metabolomic analysis of patients with <Emphasis Type="Italic">Clostridium difficile</Emphasis> infection: a pilot study

Introduction

Recurrent Clostridium difficile infection (CDI) is associated with intestinal dysbiosis. Currently, there is no diagnostic test to predict at-risk patients for CDI recurrences. Urine metabolomics may have prognostic value, but have not been characterized in this patient population.

Objective

The aim of this pilot study was to profile the urine metabolomics of patients with various frequencies of CDI.

Methods

Spot urine samples were prospectively collected from 31 adults who at various stages of recurrent CDI (1 to >5 episodes). Patients were age- and sex-matched in a 1:1 ratio with healthy controls. Urine metabolomics was performed and spectra were assessed using Chenomx NMRSuite v7 and analyzed using multivariate statistics with MetaboAnalyst 3.0. Stool metagenomic analyses were performed in six patients with >3 episodes of CDI and compared to 7 healthy controls, which were correlated with urine metabolomics.

Results

Using 53 metabolites, a two-component, partial least squares—discriminant analysis (PLS-DA) was built that clearly discriminated between healthy controls and CDI patients. The anticipated gender-based difference was not found within the CDI patient group. However, separations between (1) healthy control and CDI patients, as well as (2) patients with different episodes of CDI were possible and the permutations found were significant. Furthermore, choline was found to be the single most important urine metabolite separating healthy controls from CDI patients, and the microbiota from recurrent CDI patients was found to have decreased abundance of choline metabolizing bacteria.

Conclusions

Using small groups in a preliminary study, we have demonstrated that urine metabolomics has the potential to distinguish between healthy controls and patients with CDI. Furthermore, it could discriminate between patients experiencing different frequencies of recurrent CDI. If validated in larger cohorts, urine metabolomics has potential at identifying patients who are at risk for recurrent CDI. The significance of choline-deficient microbiota in CDI patients should be further examined.

     

FSH receptors in the bovine corpus luteum

Binding of follicle stimulating hormone (FSH) to a crude membrane fraction of bovine corpus luteum (CL) has been detected. This binding meets the usual criteria for a receptor based on specificity, time course of reaction and association constant (Ka = 8.5 x 10(10)M(-1)). Physiological studies with CL removed from heifers at specific times after estrus indicate that day-6 CL had the highest FSH binding. However, a correlation with physiological function was not obvious since some functional mid-cycle CL were high in progesterone and luteinizing hormone (LH) receptor but had nondetectable FSH receptor. Conversely, some late-cycle CL had low progesterone and LH receptor but significant quantities of FSH receptor.     

High order quaternary arrangement confers increased structural stability to Brucella sp. lumazine synthase

The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18-kDa monomers, each extensively contacting neighboring monomers. The icosahedrical structure consists of 60 LS monomers arranged as 12 pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella sp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedric enzymes. This unusual divergence prompted us to further investigate its quaternary arrangement. In the present work, we demonstrate by means of solution light scattering and x-ray structural analyses that BLS assembles as a very stable dimer of pentamers, representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 degrees C compared with pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins.     

Neonatal estrogen exposure of male rats alters reproductive functions at adulthood

The effects of neonatal exposure to different doses of diethylstilbestrol (DES) on the reproductive functions of male rats at adulthood were evaluated. Sprague-Dawley rats (5-8/group) received sc injections of 25 microl olive oil containing DES (Sigma Chemical Co., St. Louis, MO) at a dose of 10 microg, 1 microg, 100 ng, 10 ng, or 1 ng per rat on alternate days from Postnatal Days 2-12. Control animals received olive oil only. All animals were allowed to develop until 83-91 days of age; however, when they were 70 to 80 days old, four male rats each from the 10 microg, 1 microg, 100 ng, and control groups were cohabited with untreated 60- to 70-day-old females (1:1) for 12 days. At the end of cohabitation, both mated and unmated male rats were weighed, and blood and tissue samples were collected and processed. Results revealed that although sperm motility patterns and sperm morphology were adversely affected in the 10- microg group, other reproductive parameters, including 1). daily sperm production (DSP)/testis; 2). absolute and relative weights of the testis, epididymis, and seminal vesicle; and 3). sperm numbers in both regions of the epididymis declined significantly in a dose-dependent manner in the 10- and 1- microg groups. Conversely, in the <1- microg groups, none of these parameters (except DSP/testis and weight of the epididymis in the 100-ng group, and sperm numbers in the epididymis of the 100- and 10-ng groups) was different from controls. Generally, plasma testosterone levels decreased in the 10- and 1- microg groups, FSH level increased in the 10-microg group, and prolactin and LH levels were unaltered. In the fertility study, although each male in the 1-microg, 100-ng, and control groups produced a copulatory plug and impregnated a female, none could do so in the 10-microg group. The mean number of pups per litter was reduced to eight in the 1-microg group, in contrast to 15 each in the 100-ng and control groups. In conclusion, exposure of neonatal male rats to DES altered sperm motility patterns, sperm fertility (as evident from the reduced number of pups in the 1-microg group), and sexual behavior (as evident from the absence of copulatory plugs in the 10-microg group) and reduced weights of reproductive organs, DSP/testis, and sperm numbers in the epididymis. Whether these alterations/reductions persist in older rats (6-8 mo of age) is under investigation.     

Cellular spin resonance of aging yeast and mouse sarcoma cells

The way in which individual cells may be made to spin by the application of alternating electric fields is examined. The spinning of a given living cell is observed to respond rather sharply and in a resonant manner at several frequencies, hence the term "cellular spin resonance" (CSR). The frequencies of the applied field can be orders of magnitude higher than the actual spin rate. The CSR varies with the conductivity of the medium, with the square of the applied field, with the cell type and with the phase of the yeast cell life cycle. Living cells respond readily and individually are sharply resonant. Dead cells show little such response.From the behavior of the CRS in sinusoidal AC, as compared to pulsed DC, it appears likely that one cause of CSR, at least that in high frequency electric fields, is the presence of natural rf oscillations arising from the cells, and modulated by their high polarizability.     

Three-dimensional structure of the Fab from a human IgM cold agglutinin

Cold agglutinins (CAs) are IgM autoantibodies characterized by their ability to agglutinate in vitro RBC at low temperatures. These autoantibodies cause hemolytic anemia in patients with CA disease. Many diverse Ags are recognized by CAs, most frequently those belonging to the I/i system. These are oligosaccharides composed of repeated units of N:-acetyllactosamine, expressed on RBC. The three-dimensional structure of the Fab of KAU, a human monoclonal IgM CA with anti-I activity, was determined. The KAU combining site shows an extended cavity and a neighboring pocket. Residues from the hypervariable loops V(H)CDR3, V(L)CDR1, and V(L)CDR3 form the cavity, whereas the small pocket is defined essentially by residues from the hypervariable loops V(H)CDR1 and V(H)CDR2. This fact could explain the V(H)4-34 germline gene restriction among CA. The KAU combining site topography is consistent with one that binds a polysaccharide. The combining site overall dimensions are 15 A wide and 24 A long. Conservation of key binding site residues among anti-I/i CAs indicates that this is a common feature of this family of autoantibodies. We also describe the first high resolution structure of the human IgM C(H)1:C(L) domain. The structural analysis shows that the C(H)1-C(L) interface is mainly conserved during the isotype switch process from IgM to IgG1.