NMR analysis of the CSF and plasma metabolome of rigorously matched amyotrophic lateral sclerosis,Parkinson’s disease and control subjects

Introduction

Amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) are two severe neurodegenerative disorders for which the disease mechanisms are poorly understood and reliable biomarkers are absent.

Objectives

To identify metabolite biomarkers for ALS and PD, and to gain insights into which metabolic pathways are involved in disease.

Methods

Nuclear magnetic resonance (NMR) metabolomics was utilized to characterize the metabolite profiles of cerebrospinal fluid (CSF) and plasma from individuals in three age, gender, and sampling-date matched groups, comprising 22 ALS, 22 PD and 28 control subjects.

Results

Multivariate analysis of NMR data generated robust discriminatory models for separation of ALS from control subjects. ALS patients showed increased concentrations of several metabolites in both CSF and plasma, these are alanine (CSF fold change = 1.22, p = 0.005), creatine (CSF-fc = 1.17, p = 0.001), glucose (CSF-fc = 1.11, p = 0.036), isoleucine (CSF-fc = 1.24, p = 0.002), and valine (CSF-fc = 1.17, p = 0.014). Additional metabolites in CSF (creatinine, dimethylamine and lactic acid) and plasma (acetic acid, glutamic acid, histidine, leucine, pyruvate and tyrosine) were also important for this discrimination. Similarly, panels of CSF-metabolites that discriminate PD from ALS and control subjects were identified.

Conclusions

The results for the ALS patients suggest an affected creatine/creatinine pathway and an altered branched chain amino acid (BCAA) metabolism, and suggest links to glucose and energy metabolism. Putative metabolic markers specific for ALS (e.g. creatinine and lactic acid) and PD (e.g. 3-hydroxyisovaleric acid and mannose) were identified, while several (e.g. creatine and BCAAs) were shared between ALS and PD, suggesting some overlap in metabolic alterations in these disorders.

     

Human-induced Trophic Cascades and Ecological Regime Shifts in the Baltic Sea

Abstract The ecosystems of coastal and enclosed seas are under increasing anthropogenic pressure worldwide, with Chesapeake Bay, the Gulf of Mexico and the Black and Baltic Seas as well known examples. We use an ecosystem model (Ecopath with Ecosim, EwE) to show that reduced top-down control (seal predation) and increased bottom-up forcing (eutrophication) can largely explain the historical dynamics of the main fish stocks (cod, herring and sprat) in the Baltic Sea between 1900 and 1980. Based on these results and the historical fish stock development we identify two major ecological transitions. A shift from seal to cod domination was caused by a virtual elimination of marine mammals followed by a shift from an oligotrophic to a eutrophic state. A third shift from cod to clupeid domination in the late 1980s has previously been explained by overfishing of cod and climatic changes. We propose that the shift from an oligotrophic to a eutrophic state represents a true regime shift with a stabilizing mechanism for a hysteresis phenomenon. There are also mechanisms that could stabilize the shift from a cod to clupeid dominated ecosystem, but there are no indications that the ecosystem has been pushed that far yet. We argue that the shifts in the Baltic Sea are a consequence of human impacts, although variations in climate may have influenced their timing, magnitude and persistence. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fragmentation heterogeneity of 23S ribosomal RNA in Haemophilus species

The fragmentation of 23S rRNA of 22 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112 bp long intervening sequence (IVS) at position 542 and a conserved 121–123 bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria.     

Symbionts as major modulators of insect health: lactic acid bacteria and honeybees

Lactic acid bacteria (LAB) are well recognized beneficial host-associated members of the microbiota of humans and animals. Yet LAB-associations of invertebrates have been poorly characterized and their functions remain obscure. Here we show that honeybees possess an abundant, diverse and ancient LAB microbiota in their honey crop with beneficial effects for bee health, defending them against microbial threats. Our studies of LAB in all extant honeybee species plus related apid bees reveal one of the largest collections of novel species from the genera Lactobacillus and Bifidobacterium ever discovered within a single insect and suggest a long (>80 mya) history of association. Bee associated microbiotas highlight Lactobacillus kunkeei as the dominant LAB member. Those showing potent antimicrobial properties are acquired by callow honey bee workers from nestmates and maintained within the crop in biofilms, though beekeeping management practices can negatively impact this microbiota. Prophylactic practices that enhance LAB, or supplementary feeding of LAB, may serve in integrated approaches to sustainable pollinator service provision. We anticipate this microbiota will become central to studies on honeybee health, including colony collapse disorder, and act as an exemplar case of insect-microbe symbiosis.     

Deformed wing virus associated with <Emphasis Type="Italic">Tropilaelaps mercedesae</Emphasis> infesting European honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Mites in the genus Tropilaelaps (Acari: Laelapidae) are ectoparasites of the brood of honey bees (Apis spp.). Different Tropilaelaps subspecies were originally described from Apis dorsata, but a host switch occurred to the Western honey bee, Apis mellifera, for which infestations can rapidly lead to colony death. Tropilaelaps is hence considered more dangerous to A. mellifera than the parasitic mite Varroa destructor. Honey bees are also infected by many different viruses, some of them associated with and vectored by V. destructor. In recent years, deformed wing virus (DWV) has become the most prevalent virus infection in honey bees associated with V. destructor. DWV is distributed world-wide, and found wherever the Varroa mite is found, although low levels of the virus can also be found in Varroa free colonies. The Varroa mite transmits viral particles when feeding on the haemolymph of pupae or adult bees. Both the Tropilaelaps mite and the Varroa mite feed on honey bee brood, but no observations of DWV in Tropilaelaps have so far been reported. In this study, quantitative real-time RT-PCR was used to show the presence of DWV in infested brood and Tropilaelaps mercedesae mites collected in China, and to demonstrate a close quantitative association between mite-infested pupae of A. mellifera and DWV infections. Phylogenetic analysis of the DWV sequences recovered from matching pupae and mites revealed considerable DWV sequence heterogeneity and polymorphism. These polymorphisms appeared to be associated with the individual brood cell, rather than with a particular host.     

Studies on the distribution of calcitonin gene-related peptide-like and substance P-like immunoreactivities in rat hind limb muscles

Summary The tibialis anterior, extensor digitorum longus and soleus muscles in the rat were examined with respect to the presence of calcitonin gene-related peptide-like as well as substance P-like immunoreactivity. In some of the motor endplates in these muscles, identified by staining for acetylcholinesterase activity, calcitonin gene-related peptide-like immunoreactivity was detected, but in others it was not. Calcitonin gene-related peptide-like immunoreactivity was found to coexist with substance-P-like immunoreactivity in nerve fibres located outside and inside the capsule of the muscle spindles, as well as in nerve fibres located in nerve fascicles. These fibres presumably represent sensory nerve fibres. Calcitonin gene-related peptide-like immunoreactivity, but not substance P-like immunoreactivity, was also detected, in cap-like structures located on the surface of the intrafusal muscle fibres in the polar regions of the spindles, structures which are likely to correspond to motor plate endings. The observations suggest that calcitonin gene-related peptide is heterogeneously present in the endplates of rat hind limb muscles, and gives for the first time immunohistochemical evidence for the presence of calcitonin gene-related peptide and substance P in the innervation of muscle spindles.     

Pronounced substance P innervation in irradiation-induced enteropathy--a study on human colon

The immunohistochemical expression of various neuropeptides, including substance P (SP), and the substance P receptor (SPR), was examined in irradiation-induced enteropathy in man. Samples from irradiated and non-irradiated patients operated on for rectal carcinoma were examined. The samples were from the sigmoid and corresponded macroscopically to non-cancerous sigmoid colon. There was a marked atrophy, ulcerations and inflammatory reactions in the irradiation-influenced mucosa. In this mucosa, there was a very pronounced innervation of varicose nerve fibers showing SP-like immunoreactivity (LI). The degree of SP-LI in the ganglionic cells of the submucous plexus was increased as compared to non-irradiated patients. There were only few or no nerve fibers showing immunoreaction for other neuropeptides examined (CGRP, enkephalin, NPY) in the irradiation-influenced mucosa. A marked SPR immunoreaction was detected in cells in the lamina propria which were interpreted as representing polymorphonuclear leukocytes. The marked expression of SP in the irradiation-damaged mucosa and the presence of SPR immunoreactive leukocytes suggest that SP is highly involved in the inflammatory reactions that occur in response to radiotherapy. The observations also suggest that SP, but not NPY, CGRP and enkephalin, has an important role in the reorganisation processes that take place in the mucosa in irradiation-induced enteropathy.     

Development and evaluation of a core genome multilocus sequence typing scheme for Paenibacillus larvae,the deadly American foulbrood pathogen of honeybees

Paenibacillus larvae is the causative agent of the fatal American foulbrood disease in honeybees (Apis mellifera). Strain identification is vital for preventing the spread of the disease. To date, the most accessible and robust scheme to identify strains is the multilocus sequence typing (MLST) method. However, this approach has limited resolution, especially for epidemiological studies. As the cost of whole-genome sequencing has decreased and as it becomes increasingly available to most laboratories, an extended MLST based on the core genome (cgMLST) presents a valuable tool for high-resolution investigations. In this study, we present a standardized, robust cgMLST scheme for P. larvae typing using whole-genome sequencing. A total of 333 genomes were used to identify, validate and evaluate 2419 core genes. The cgMLST allowed fine-scale differentiation between samples that had the same profile using traditional MLST and allowed for the characterization of strains impossible by MLST. The scheme was successfully used to trace a localized Swedish outbreak, where a cluster of 38 isolates was linked to a country-wide beekeeping operation. cgMLST greatly enhances the power of a traditional typing scheme, while preserving the same stability and standardization for sharing results and methods across different laboratories.     

Effect of diabetic state on co-localization of substance P and serotonin in the gut in animal models

Changes in the numbers of serotonin- and substance P-immunoreactive (IR) cells occur in several animal models of diabetes. It is not known, however, whether these changes are a result of actual cell loss or are caused by modified gene expression in cells showing co-localization of serotonin and substance P. The pattern of mono- and co-expression of serotonin, as well as of substance P, was therefore investigated in gastrointestinal endocrine cells from animal models of human type 1 and type 2 diabetes, namely non-obese diabetic (NOD) and obese diabetic (ob/ob) mice. Immunocytochemical staining by the avidin-biotin complex method was performed for computerized image analysis of each cell type, and by immunofluorescence double staining to study co-localization. Tissues from antrum, proximal duodenum and distal colon were investigated. Co-localization of serotonin- and substance P-IR was found in all investigated parts of the gut. In antrum, substance P immunoreactivity was found exclusively in serotonin-IR cells. In both NOD and ob/ob mice there was a reduced number of substance P-IR cells, but an unchanged serotonin-IR cell count, which thus tallies with a shut-off of substance P expression in antral enterochromaffin cells. In duodenum, both diabetes models showed a decreased number of serotonin-IR cells. Furthermore there was a decreased number of substance P-IR cells in the type 2 model. The proportion of serotonin-IR cells showing substance P-immuno-reactivity was decreased in both diabetic models, thus indicating a shut-off of substance P-gene expression. However, this does not fully explain the changes in duodenum, but the diabetic state probably affects the number of mono-expressed cells as well. In colon, no change was found in diabetic mice regarding co-localization of substance P and serotonin. However, pre-diabetic NOD mice showed a decreased proportion of substance P in serotonin-IR cells, which might be explained by the increased number of serotonin-IR cells, combined with an unchanged number of substance P-IR cells. In conclusion, diabetic animal models of both type 1 and type 2 appear to have a combination of decreased expression of substance P in serotonin-IR cells of both antrum and duodenum, as well as a change in the number of mono-expressed cells. The pattern in colon, on the other hand, seems to be unaffected.     

Nerve Cells Associated with the Endocrine Pancreas in Young Mice: An Ultrastructural Analysis of the Neuroinsular Complex Type I

The neuroinsular complex type 1 is composed of pancreatic endocrine islet cells and nerve cell bodies intrinsic to the islet. The details of the relation between nerve cells and between endocrine cells and nerve cells in the complex are unknown. Pancreata from newborn and 18-day-old mice were analysed by electron microscopy to establish the ultrastructural morphology of the neuroinsular complex. Immunohistochemical staining for protein gene-product 9.5 was also performed. The study showed that nerve cell bodies were closely associated to each other in the periphery of the islets with no connective tissue separating the cells. The nerve cells were closely associated to both -cells and -cells. Direct intercellular contacts were observed between nerve cells and endocrine cells and between Schwann cells and endocrine cells. Varicose nerve endings were frequently observed in the neuroinsular complex. In the peripheral parts the varicosities were mostly being associated to the nerve cell bodies. The varicosities contained small clear or small clear and larger dense cored vesicles, suggesting cholinergic and peptidergic contents. The varicosities made specialized synaptic connections with adjacently located nerve cells. The study shows that the neuronal part of the neuroinsular complex is closely associated to the endocrine islet cells and that it is richly innervated, indicating an important regulatory function of the nerve cell component in the neuroinsular complex.