Next-Generation Sequencing of the Human Aqueous Humour Microbiome.

The microbiome of the ocular surface has been characterised, but only limited information is available on a possible silent intraocular microbial colonisation in normal eyes. Therefore, we performed next-generation sequencing (NGS) of 16S rDNA genes in the aqueous humour. The aqueous humour was sampled from three patients during cataract surgery. Air swabs, conjunctival swabs from patients as well as from healthy donors served as controls. Following DNA extraction, the V3 and V4 hypervariable regions of the 16S rDNA gene were amplified and sequenced followed by denoising. The resulting Amplicon Sequence Variants were matched to a subset of the Ribosomal Database Project 16S database. The deduced bacterial community was then statistically analysed. The DNA content in all samples was low (0-1.49 ng/µL) but sufficient for analysis. The main phyla in the samples were Acinetobacteria (48%), Proteobacteria (26%), Firmicutes (14%), Acidobacteria (8%), and Bacteroidetes (2%). Patients' conjunctival control samples and anterior chamber fluid showed similar patterns of bacterial species containing many waterborne species. Non-disinfected samples showed a different bacterial spectrum than the air swab samples. The data confirm the existence of an ocular surface microbiome. Meanwhile, a distinct intraocular microbiome was not discernible from the background, suggesting the absence of an intraocular microbiome in normal eyes.

Nup133 and ERα mediate the differential effects of hyperoxia-induced damage in male and female OPCs.

BACKGROUND: Hyperoxia is a well-known cause of cerebral white matter injury in preterm infants with male sex being an independent and critical risk factor for poor neurodevelopmental outcome. Sex is therefore being widely considered as one of the major decisive factors for prognosis and treatment of these infants. But unfortunately, we still lack a clear view of the molecular mechanisms that lead to such a profound difference. Hence, using mouse-derived primary oligodendrocyte progenitor cells (OPCs), we investigated the molecular factors and underlying mechanisms behind the differential response of male and female cells towards oxidative stress. RESULTS: We demonstrate that oxidative stress severely affects cellular functions related to energy metabolism, stress response, and maturation in the male-derived OPCs, whereas the female cells remain largely unaffected. CNPase protein level was found to decline following hyperoxia in male but not in female cells. This impairment of maturation was accompanied by the downregulation of nucleoporin and nuclear lamina proteins in the male cells. We identify Nup133 as a novel target protein affected by hyperoxia, whose inverse regulation may mediate this differential response in the male and female cells. Nup133 protein level declined following hyperoxia in male but not in female cells. We show that nuclear respiratory factor 1 (Nrf1) is a direct downstream target of Nup133 and that Nrf1 mRNA declines following hyperoxia in male but not in female cells. The female cells may be rendered resistant due to synergistic protection via the estrogen receptor alpha (ERα) which was upregulated following hyperoxia in female but not in male cells. Both Nup133 and ERα regulate mitochondrial function and oxidative stress response by transcriptional regulation of Nrf1. CONCLUSIONS: These findings from a basic cell culture model establish prominent sex-based differences and suggest a novel mechanism involved in the differential response of OPCs towards oxidative stress. It conveys a strong message supporting the need to study how complex cellular processes are regulated differently in male and female brains during development and for a better understanding of how the brain copes up with different forms of stress after preterm birth.

Regulation of hepatic microRNAs in response to early stage Echinococcus multilocularis egg infection in C57BL/6 mice.

We present a comprehensive analysis of the hepatic miRNA transcriptome at one month post-infection of experimental primary alveolar echinococcosis (AE), a parasitic infection caused upon ingestion of E. multilocularis eggs. Liver tissues were collected from infected and non-infected C57BL/6 mice, then small RNA libraries were prepared for next-generation sequencing (NGS). We conducted a Stem-loop RT-qPCR for validation of most dysregulated miRNAs. In infected mice, the expression levels of 28 miRNAs were significantly altered. Of these, 9 were up-regulated (fold change (FC) ≥ 1.5) and 19 were down-regulated (FC ≤ 0.66) as compared to the non-infected controls. In infected livers, mmu-miR-148a-3p and mmu-miR-101b-3p were 8- and 6-fold down-regulated, respectively, and the expression of mmu-miR-22-3p was reduced by 50%, compared to non-infected liver tissue. Conversely, significantly higher hepatic levels were noted for Mus musculus (mmu)-miR-21a-5p (FC = 2.3) and mmu-miR-122-5p (FC = 1.8). In addition, the relative mRNA expression levels of five genes (vegfa, mtor, hif1-α, fasn and acsl1) that were identified as targets of down-regulated miRNAs were significantly enhanced. All the five genes exhibited a higher expression level in livers of E. multilocularis infected mice compared to non-infected mice. Finally, we studied the issue related to functionally mature arm selection preference (5p and/or 3p) from the miRNA precursor and showed that 9 pre-miRNAs exhibited different arm selection preferences in normal versus infected liver tissues. In conclusion, this study provides first evidence that miRNAs are regulated early in primary murine AE. Our findings raise intriguing questions such as (i) how E. multilocularis affects hepatic miRNA expression;(ii) what are the alterations in miRNA expression patterns in more advanced AE-stages; and (iii) which hepatic cellular, metabolic and/or immunologic processes are modulated through altered miRNAs in AE. Thus, further research on the regulation of miRNAs during AE is needed, since miRNAs constitute an attractive potential option for development of novel therapeutic approaches against AE.

Encrustations on ureteral stents from patients without urinary tract infection reveal distinct urotypes and a low bacterial load.

BACKGROUND: Current knowledge of the urinary tract microbiome is limited to urine analysis and analysis of biofilms formed on Foley catheters. Bacterial biofilms on ureteral stents have rarely been investigated, and no cultivation-independent data are available on the microbiome of the encrustations on the stents. RESULTS: The typical encrustations of organic and inorganic urine-derived material, including microbial biofilms formed during 3-6 weeks on ureteral stents in patients treated for kidney and ureteral stones, and without reported urinary tract infection at the time of stent insertion, were analysed. Next-generation sequencing of the 16S rRNA gene V3-V4 region revealed presence of different urotypes, distinct bacterial communities. Analysis of bacterial load was performed by combining quantification of 16S rRNA gene copy numbers by qPCR with microscopy and cultivation-dependent analysis methods, which revealed that ureteral stent biofilms mostly contain low numbers of bacteria. Fluorescence microscopy indicates the presence of extracellular DNA. Bacteria identified in biofilms by microscopy had mostly morphogenic similarities to gram-positive bacteria, in few cases to Lactobacillus and Corynebacterium, while sequencing showed many additional bacterial genera. Weddellite crystals were absent in biofilms of patients with Enterobacterales and Corynebacterium-dominated microbiomes. CONCLUSIONS: This study provides novel insights into the bacterial burden in ureteral stent encrustations and the urinary tract microbiome. Short-term (3-6 weeks) ureteral stenting is associated with a low load of viable and visible bacteria in ureteral stent encrustations, which may be different from long-term stenting. Patients could be classified according to different urotypes, some of which were dominated by potentially pathogenic species. Facultative pathogens however appear to be a common feature in patients without clinically manifested urinary tract infection. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02845726 . Registered on 30 June 2016-retrospectively registered.

HHV-6 encoded small non-coding RNAs define an intermediate and early stage in viral reactivation.

Human herpesvirus 6A and 6B frequently acquires latency. HHV-6 activation has been associated with various human diseases. Germ line inheritance of chromosomally integrated HHV-6 makes viral DNA-based analysis difficult for determination of early stages of viral activation. We characterized early stages of HHV-6 activation using high throughput transcriptomics studies and applied the results to understand virus activation under clinical conditions. Using a latent HHV-6A cell culture model in U2OS cells, we identified an early stage of viral reactivation, which we define as transactivation that is marked by transcription of several viral small non-coding RNAs (sncRNAs) in the absence of detectable increase in viral replication and proteome. Using deep sequencing approaches, we detected previously known as well as a new viral sncRNAs that characterized viral transactivation and differentiated it from latency. Here we show changes in human transcriptome upon viral transactivation that reflect multiple alterations in mitochondria-associated pathways, which was supported by observation of increased mitochondrial fragmentation in virus reactivated cells. Furthermore, we present here a unique clinical case of DIHS/DRESS associated death where HHV-6 sncRNA-U14 was abundantly detected throughout the body of the patient in the presence of low viral DNA. In this study, we have identified a unique and early stage of viral activation that is characterized by abundant transcription of viral sncRNAs, which can serve as an ideal biomarker under clinical conditions.

PBT assessment under REACH: Screening for low aquatic bioaccumulation with QSAR classifications based on physicochemical properties to replace BCF in vivo testing on fish.

Aquatic bioconcentration factors (BCFs) are critical in PBT (persistent, bioaccumulative, toxic) and risk assessment of chemicals. High costs and use of more than 100 fish per standard BCF study (OECD 305) call for alternative methods to replace as much in vivo testing as possible. The BCF waiving scheme is a screening tool combining QSAR classifications based on physicochemical properties related to the distribution (hydrophobicity, ionisation), persistence (biodegradability, hydrolysis), solubility and volatility (Henry's law constant) of substances in water bodies and aquatic biota to predict substances with low aquatic bioaccumulation (nonB, BCF<2000). The BCF waiving scheme was developed with a dataset of reliable BCFs for 998 compounds and externally validated with another 181 substances. It performs with 100% sensitivity (no false negatives), >50% efficacy (waiving potential), and complies with the OECD principles for valid QSARs. The chemical applicability domain of the BCF waiving scheme is given by the structures of the training set, with some compound classes explicitly excluded like organometallics, poly- and perfluorinated compounds, aromatic triphenylphosphates, surfactants. The prediction confidence of the BCF waiving scheme is based on applicability domain compliance, consensus modelling, and the structural similarity with known nonB and B/vB substances. Compounds classified as nonB by the BCF waiving scheme are candidates for waiving of BCF in vivo testing on fish due to low concern with regard to the B criterion. The BCF waiving scheme supports the 3Rs with a possible reduction of >50% of BCF in vivo testing on fish. If the target chemical is outside the applicability domain of the BCF waiving scheme or not classified as nonB, further assessments with in silico, in vitro or in vivo methods are necessary to either confirm or reject bioaccumulative behaviour.

A comparative survey of chemistry-driven in silico methods to identify hazardous substances under REACH.

This paper presents an inventory of in silico screening tools to identify substance properties of concern under the European chemicals' legislation REACH. The objective is to support the selection and implementation of appropriate tools as building blocks within integrated testing strategies (ITS). The relevant concerns addressed are persistence, bioaccumulation potential, acute and long-term aquatic toxicity, PBT/vPvB properties ((very) persistent, (very) bioaccumulative, toxic), CMR (carcinogenicity, mutagenicity, reproductive toxicity), endocrine disruption and skin sensitisation. The inventory offers a comparative evaluation of methods with respect to the underlying algorithms (how does the method work?) and the applicability domains (when does the method work?) as well as their limitations (when does the method not work?). The inventory explicitly addresses the reliability of predictions of different in silico models for diverse chemicals by applicability domain considerations. The confidence in predictions can be greatly improved by consensus modelling that allows for taking conflicting results into account. The inventory is complemented by a brief discussion of socio-economic tools for assessing the potential efficiency gains of using in silico methods compared to traditional in vivo testing of chemical hazards.

Using conditional inference trees and random forests to predict the bioaccumulation potential of organic chemicals.

The present study presents a data-oriented, tiered approach to assessing the bioaccumulation potential of chemicals according to the European chemicals regulation on Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). The authors compiled data for eight physicochemical descriptors (partition coefficients, degradation half-lives, polarity, and so forth) for a set of 713 organic chemicals for which experimental values of the bioconcentration factor (BCF) are available. The authors employed supervised machine learning methods (conditional inference trees and random forests) to derive relationships between the physicochemical descriptors and the BCF values. In a first tier, the authors established rules for classifying a chemical as bioaccumulative (B) or nonbioaccumulative (non-B). In a second tier, the authors developed a new tool for estimating numerical BCF values. For both cases the optimal set of relevant descriptors was determined; these are biotransformation half-life and octanol-water distribution coefficient (log D) for the classification rules and log D, biotransformation half-life, and topological polar surface area for the BCF estimation tool. The uncertainty of the BCF estimates obtained with the new estimation tool was quantified by comparing the estimated and experimental BCF values of the 713 chemicals. Comparison with existing BCF estimation methods indicates that the performance of this new BCF estimation tool is at least as high as that of existing methods. The authors recommend the present study's classification rules and BCF estimation tool for a consensus application in combination with existing BCF estimation methods.

Screening for PBT chemicals among the "existing" and "new" chemicals of the EU.

Under the European chemicals legislation, REACH, industrial chemicals that are imported or manufactured at more than 10 t/yr need to be evaluated with respect to their persistence (P), bioaccumulation potential (B), and toxicity (T). This assessment has to be conducted for several 10,000 of chemicals but, at the same time, empirical data on degradability, bioaccumulation potential and toxicity of industrial chemicals are still scarce. Therefore, the identification of PBT chemicals among all chemicals on the market remains a challenge. We present a PBT screening of approximately 95,000 chemicals based on a comparison of estimated P, B, and T properties of each chemical with the P, B, and T thresholds defined under REACH. We also apply this screening procedure to a set of 2576 high production volume chemicals and a set of 2781 chemicals from the EU's former list of "new chemicals" (ELINCS). In the set of 95,000 chemicals, the fraction of potential PBT chemicals is around 3%, but in the ELINCS chemicals it reaches 5%. We identify the most common structural elements among the potential PBT chemicals. Analysis of the P, B, and T data for all chemicals considered here shows that the uncertainty in persistence data contributes most to the uncertainty in the number of potential PBT chemicals.

Norepinephrine transporter inhibition alters the hemodynamic response to hypergravitation.

Sympathetically mediated tachycardia and vasoconstriction maintain blood pressure during hypergravitational stress, thereby preventing gravitation-induced loss of consciousness. Norepinephrine transporter (NET) inhibition prevents neurally mediated (pre)syncope during gravitational stress imposed by head-up tilt testing. Thus it seems reasonable that NET inhibition could increase tolerance to hypergravitational stress. We performed a double-blind, randomized, placebo-controlled crossover study in 11 healthy men (26 +/- 1 yr, body mass index 24 +/- 1 kg/m2), who ingested the selective NET inhibitor reboxetine (4 mg) or matching placebo 25, 13, and 1 h before testing on separate days. We monitored heart rate, blood pressure, and thoracic impedance in three different body positions (supine, seated, standing) and during a graded centrifuge run (incremental steps of 0.5 g for 3 min each, up to a maximal vertical acceleration load of 3 g). NET inhibition increased supine blood pressure and heart rate. With placebo, blood pressure increased in the seated position and was well maintained during standing. However, with NET inhibition, blood pressure decreased in the seated and standing position. During hypergravitation, blood pressure increased in a graded fashion with placebo. With NET inhibition, the increase in blood pressure during hypergravitation was profoundly diminished. Conversely, the tachycardic responses to sitting, standing, and hypergravitation all were greatly increased with NET inhibition. In contrast to our expectation, short-term NET inhibition did not improve tolerance to hypergravitation. Redistribution of sympathetic activity to the heart or changes in baroreflex responses could explain the excessive tachycardia that we observed.