First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid- chromatography–Urgent need for harmonisation of analytical methods

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs

Subtle changes in striatal muscarinic M1 and M4 receptor expression in the DYT1 knock-in mouse model of dystonia.

In early-onset generalized torsion dystonia, caused by a GAG deletion in TOR1A (DYT1), enhanced striatal cholinergic activity has been suggested to be critically involved. Previous studies have shown increased acetylcholine levels in the striatum of DYT1 knock-in (KI) mice. Ex vivo data indicated that muscarinic receptor antagonists normalize the activity of striatal cholinergic interneurons. Currently receptor subtype specific antagonists are developed for therapy, however, it is yet unknown whether the levels of targeted receptors are unaltered. In the present study, we firstly examined the expression of M1 and M4 receptors in DYT1 KI mice in comparison to wildtype mice. While no changes in mRNA were found in the motor cortex, the expression of M1 was higher in the striatum of DYT1 KI. However, M1 protein did not differ in striatum and cortex between the animal groups as shown by immunohistochemistry and western blot. M4 receptor protein, unaltered in the cortex, was slightly lower in lateral subparts of the striatum, but unchanged in somata of cholinergic interneurons and substance P immunoreactive projection neurons. Functional alterations of the cholinergic system and of aberrant striatal plasticity, demonstrated by previous studies, seem not to be related to overt changes in M1 and M4 expression. This critically informs the ongoing development of respective antagonists for therapy of dystonia

Polyethylenimine Nanoparticle-Mediated siRNA Delivery to Reduce α-Synuclein Expression in a Model of Parkinson’s Disease

RNA interference (RNAi)-based strategies that mediate the specific knockdown of target genes by administration of small interfering RNAs (siRNAs) could be applied for treatment of presently incurable neurodegenerative diseases such as Parkinson’s disease. However, inefficient delivery of siRNA into neurons hampers in vivo application of RNAi. We have previously established the 4–12 kDa branched polyethylenimine (PEI) F25-LMW with superior transfection efficacy for delivery of siRNA in vivo. Here, we present that siRNA complexed with this PEI extensively distributes across the CNS down to the lumbar spinal cord after a single intracerebroventricular infusion. siRNA against α-synuclein (SNCA), a pre-synaptic protein that aggregates in Parkinson’s disease, was complexed with PEI F25-LMW and injected into the lateral ventricle of mice overexpressing human wild-type SNCA (Thy1-aSyn mice). Five days after the single injection of 0.75 μg PEI/siRNA, SNCA mRNA expression in the striatum was reduced by 65%, accompanied by reduction of SNCA protein by ∼50%. Mice did not show signs of toxicity or adverse effects. Moreover, ependymocytes and brain parenchyma were completely preserved and free of immune cell invasion, astrogliosis, or microglial activation. Our results support the efficacy and safety of PEI nanoparticle-mediated delivery of siRNA to the brain for therapeutic intervention

Flow chart of the analytic approach to identify lineage-specific biomarker levels.

<p><b>A:</b> differences in biomarker levels were sorted as specific to (i) humans, (ii) great apes, (iii) bonobos, (iv) chimpanzees, (v) Central African chimpanzees, (vi) West African chimpanzees and as (vii) non-lineage specific. <b>B:</b> Human-specific changes were defined as significant differences to chimpanzees and bonobos taken together (but not between the latter two species) as well as to rhesus macaques (shown); and as significant differences between humans and the individual great ape species (not shown), regardless of significant differences between species born and living under different environments. Relations of species as shown in cladograms derived from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134548#pone.0134548.ref103" target="_blank">103</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134548#pone.0134548.ref104" target="_blank">104</a>].</p

Lineage-Specific Changes in Biomarkers in Great Apes and Humans

<div><p>Although human biomedical and physiological information is readily available, such information for great apes is limited. We analyzed clinical chemical biomarkers in serum samples from 277 wild- and captive-born great apes and from 312 healthy human volunteers as well as from 20 rhesus macaques. For each individual, we determined a maximum of 33 markers of heart, liver, kidney, thyroid and pancreas function, hemoglobin and lipid metabolism and one marker of inflammation. We identified biomarkers that show differences between humans and the great apes in their average level or activity. Using the rhesus macaques as an outgroup, we identified human-specific differences in the levels of bilirubin, cholinesterase and lactate dehydrogenase, and bonobo-specific differences in the level of apolipoprotein A-I. For the remaining twenty-nine biomarkers there was no evidence for lineage-specific differences. In fact, we find that many biomarkers show differences between individuals of the same species in different environments. Of the four lineage-specific biomarkers, only bilirubin showed no differences between wild- and captive-born great apes. We show that the major factor explaining the human-specific difference in bilirubin levels may be genetic. There are human-specific changes in the sequence of the promoter and the protein-coding sequence of uridine diphosphoglucuronosyltransferase 1 (UGT1A1), the enzyme that transforms bilirubin and toxic plant compounds into water-soluble, excretable metabolites. Experimental evidence that UGT1A1 is down-regulated in the human liver suggests that changes in the promoter may be responsible for the human-specific increase in bilirubin. We speculate that since cooking reduces toxic plant compounds, consumption of cooked foods, which is specific to humans, may have resulted in relaxed constraint on UGT1A1 which has in turn led to higher serum levels of bilirubin in humans.</p></div

Liver UDP-glucuronosyltransferase 1A1 (UGT1A1) promoter transcript expression in rhesus macaques, chimpanzees and humans [47] and respective TATAA-box length [56, 57, 105]

<p>- UGT1A1 transcript expression was determined from RNA-Seq of human, chimpanzee and rhesus macaque liver RNA samples from 3 males and 3 females of each species [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134548#pone.0134548.ref047" target="_blank">47</a>]. Relative expression levels were calculated from the original dataset setting human expression levels at 100 percent (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134548#pone.0134548.s007" target="_blank">S1 Table</a> for the variability of TA repeats in TATA box of UGT1A1 promoter in archaic hominins, humans and non-human primates).</p

Lineage-specific biomarker levels in humans and other primates

<p>- box representing 25th, 50th and 75th percentiles; whiskers representing 2.5^th to 97.5^th percentiles; outliers are not shown; description of species: Rh—DE: captive-born rhesus macaque samples from Germany; B—CD: wild-born bonobo samples from the Democratic Republic of the Congo; B—DE: captive-born bonobo samples from Germany; Ch—CG: wild-born Central African chimpanzee samples from the Republic of Congo; Ch—SL: wild-born West African chimpanzee samples from Sierra Leone; Ch—DE: captive-born West African chimpanzee samples Germany; H—DE: human samples from Germany; A: Bonobo-specific change in apolipoprotein A-I; human-specific change in B: bilirubin, C: cholinesterase and D: lactate; for determination of bilirubin, 1.71 μmol/L represents the lower limit of quantification of the assay.</p

Liver <i>UGT1A1</i>-mRNA expression in mice on raw and cooked diets:

<p>Liver mRNA expression of UGT1A1 transcripts in mice fed either a raw or cooked meat or raw or cooked or tuber diets was measured by RNA-Seq [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134548#pone.0134548.ref049" target="_blank">49</a>]. Total RNA was prepared from 17 individuals and sequenced as a pool on two lanes of an Illumina HiSeq 2500. Significant differences in expression between mice fed raw diets and mice fed cooked diets were quantified using DESeq [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134548#pone.0134548.ref048" target="_blank">48</a>].</p