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In contrast to bacteria, microbiome analyses often neglect archaea, but also eukaryotes. This is partly because they are difficult to culture due to their demanding growth requirements, or some even have to be classified as uncultured microorganisms. Consequently, little is known about the relevance of archaea in human health and diseases. Contemporary broad availability and spread of next generation sequencing techniques now enable a stronger focus on such microorganisms, whose cultivation is difficult. However, due to the enormous evolutionary distances between bacteria, archaea and eukaryotes, the implementation of sequencing strategies for smaller laboratory scales needs to be refined to achieve as a holistic view on the microbiome as possible. Here, we present a technical approach that enables simultaneous analyses of archaeal, bacterial and eukaryotic microbial communities to study their roles in development and courses of respiratory disorders. We thus applied combinatorial 16S-/18S-rDNA sequencing strategies for sequencing-library preparation. Considering the lower total microbiota density of airway surfaces, when compared with gut microbiota, we optimized the DNA purification workflow from nasopharyngeal swab specimens. As a result, we provide a protocol that allows the efficient combination of bacterial, archaeal, and eukaryotic libraries for nanopore-sequencing using Oxford Nanopore Technologies MinION devices and subsequent phylogenetic analyses. In a pilot study, this workflow allowed the identification of some environmental archaea, which were not correlated with airway microbial communities before. Moreover, we assessed the protocol’s broader applicability using a set of human stool samples. We conclude that the proposed protocol provides a versatile and adaptable tool for combinatorial studies on bacterial, archaeal, and eukaryotic microbiomes on a small laboratory scale.

Background: Human breast milk has a high microRNA (miRNA) content. It remains unknown whether and how milk miRNAs might affect intestinal gene regulation and homeostasis of the developing microbiome after initiating enteral nutrition. However, this requires that relevant milk miRNA amounts survive the gastrointestinal (GI) passage, are taken up by cells, and become available to the RNA interference machinery. It seems important to dissect the fate of these miRNAs after oral ingestion and GI passage. Objectives: Our goal was to analyze the potential transmissibility of milk miRNAs via the gastrointestinal system in neonate humans and a porcine model in vivo to contribute to the discussion of whether milk miRNAs could influence gene regulation in neonates and thus might vertically transmit developmental relevant signals. Methods: We performed cross-species profiling of miRNAs via deep sequencing and utilized dietary xenobiotic taxon-specific milk miRNA (xenomiRs) as tracers in human and porcine neonates, followed by functional studies in primary human fetal intestinal epithelial cells using adenovirus-type 5-mediated miRNA gene transfer. Results: Mammals share many milk miRNAs yet exhibit taxon-specific miRNA fingerprints. We traced bovine-specific miRNAs from formula nutrition in human preterm stool and 9 d after the onset of enteral feeding in intestinal cells (ICs) of preterm piglets. Thereafter, several xenomiRs accumulated in the ICs. Moreover, a few hours after introducing enteral feeding in preterm piglets with supplemented reporter miRNAs (cel-miR-39-5p/-3p), we observed their enrichment in blood serum and in argonaute RISC catalytic component 2 (AGO2)-immunocomplexes from intestinal biopsies. Conclusions: Milk-derived miRNAs survived GI passage in human and porcine neonates. Bovine-specific miRNAs accumulated in ICs of preterm piglets after enteral feeding with bovine colostrum/formula. In piglets, colostrum supplementation with cel-miR-39-5p/-3p resulted in increased blood concentrations of cel-miR-39-3p and argonaute RISC catalytic component 2 (AGO2) loading in ICs. This suggests the possibility of vertical transmission of miRNA signaling from milk through the neonatal digestive tract. Am J Clin Nutr 2023;xx:xx–xx.

We established a protocol for fast, cost efficient Sars-CoV-2 sequencing with little as possible hands-on time (around 3h in total, excluding RNA extraction). The whole Sequencing can be done in one working day, including the bioinformatic pipeline. The cost per sample accumulates at around 40$, with already isolated RNA. We adapted and simplified existing workflows using the ‘midnight’ 1,200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost all of the SARS-CoV-2 genome. Subsequently, we applied the Oxford Nanopore Rapid barcoding protocol and the portable MinION Mk1C sequencer in combination with the ARTIC bioinformatics pipeline. We tested the simplified and less time-consuming workflow on confirmed SARS-CoV-2-positive specimens from clinical routine and identified pre-analytical parameters, which may help to decrease the rate of sequencing failures. Duration of the complete pipeline was approx. 7 hrs for one specimen and approx. 11 hrs for 12 multiplexed barcoded specimens. This protocol is a modified version of Nikki Freed and Olin Silanders protocol. To get information such as Primers, visit their protocol. Nikki Freed, Olin Silander 2020. nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon).doi: 10.1093/biomethods/bpaa014 Our peer-reviewed paper is available here: https://www.mdpi.com/2076-2607/9/12/2598

BackgroundHuman breast milk has a high microRNA (miRNA) content. It remains unknown whether and how milk miRNAs might affect intestinal gene regulation and homeostasis of the developing microbiome after initiation of enteral nutrition. However, this requires that relevant milk miRNA amounts survive gastrointestinal passage, are taken up by cells, and become available to the RNA interference (RNAi) machinery. It seems important to dissect the fate of these miRNAs after oral ingestion and gastrointestinal passage.ObjectiveOur goal was to analyze the potential transmissibility of milk miRNAs via the gastrointestinal system in neonate humans and a porcine modelin vivoto contribute to the discussion whether milk miRNAs could influence gene regulation in neonates and thus might vertically transmit developmental relevant signals.DesignWe performed cross-species profiling of miRNAs via deep-sequencing and utilized dietary xenobiotic taxon-specific milk miRNA (xenomiRs) as tracers in human and porcine neonates, followed by functional studies in primary human fetal intestinal epithelial cells (HIEC-6) using Ad5-mediated miRNA-gene transfer.ResultsMammals share many milk miRNAs yet exhibit taxon-specific miRNA fingerprints. We traced bovine-specific miRNAs from formula-nutrition in human preterm stool and 9 days after onset of enteral feeding in intestinal cells of preterm piglets. Thereafter, several xenomiRs accumulated in the intestinal cells. Moreover, few hours after introducing enteral feeding in preterm piglets with supplemented reporter miRNAs (cel-miR-39-5p/-3p), we observed their enrichment in blood serum and in AGO2-immunocomplexes from intestinal biopsies.ConclusionsMilk-derived miRNAs survived gastrointestinal passage in human and porcine neonates. Bovine-specific miRNAs accumulated in intestinal cells of preterm piglets after enteral feeding with bovine colostrum/formula. In piglets, colostrum supplementation with cel-miR-39-5p/-3p resulted in increased blood levels of cel-miR-39-3p and argonaute RISC catalytic component 2 (AGO2) loading in intestinal cells. This suggests the possibility of vertical transmission of miRNA signaling from milk through the neonatal digestive tract.

Background Reverse transcription of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (+)RNA genome and subgenomic RNAs (sgRNAs) and subsequent quantitative polymerase chain reaction (RT-qPCR) is the reliable diagnostic gold standard for COVID-19 diagnosis and the identification of potential spreaders. Apart from clinical relevance and containment, for specific questions, it might be of interest to (re)investigate cases with low SARS-CoV-2 load, where RT-qPCR alone can deliver conflicting results, even though these cases might neither be clinically relevant nor significant for containment measures, because they might probably not be infectious. In order to expand the diagnostic bandwidth for non-routine questions, particularly for the reliable discrimination between negative and false-negative specimens associated with high CT values, we combined the RT-qPCR workflow with subsequent pyrosequencing of a S-gene amplicon. This expansion can help to confirm SARS-CoV-2 infections without the demand of confirmative antibody testing, which requires to summon patients again for blood sampling few to several weeks after symptom onset. Results We successfully established a combined RT-qPCR and S-gene pyrosequencing method which can be optionally exploited after routine diagnostics. This allows a reliable interpretation of RT-qPCR results in specimens with relatively low viral loads and close to the detection limits of qPCR. After laboratory implementation, we tested the combined method in a large pediatric cohort from two German medical centers (n=769). Pyrosequencing after RT-qPCR enabled us to uncover 5 previously unrecognized cases of pediatric SARS-CoV-2-associated diseases, mainly exhibiting mild and heterogeneous presentation—apart from a single case of multisystem inflammatory syndrome in children (MIS-C) associated with SARS-CoV-2, who was hospitalized in the course of the study. Conclusions The proposed protocol allows a specific and sensitive confirmation of SARS-CoV-2 infections close to the detection limits of RT-qPCR. The tested biotinylated primers do not negatively affect the RT-qPCR pipeline and thus can be optionally applied to enable deeper inspection of RT-qPCR results by subsequent pyrosequencing. Moreover, due to the incremental transmission of SARS-CoV-2 variants of concern, we note that the used strategy can uncover (Spike) P681H allowing the pre-selection of SARS-CoV-2 B.1.1.7 candidate specimens for deep sequencing.

BackgroundSurveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is essential for the global containment measures with regard to the ongoing pandemic. Diagnostic gold standard is currently reverse transcription of the (+)RNA genome and subgenomic RNAs and subsequent quantitative polymerase chain reaction (RT-qPCR) from nasopharyngeal swabs or bronchoalveolar lavages. In order to further improve the diagnostic accuracy, particularly for the reliable discrimination between negative and false-negative specimens, we propose the combination of the RT-qPCR workflow with subsequent pyrosequencing of a S-gene amplicon. This extension might add important value mainly in cases with low SARS-CoV-2 load, where RT-qPCR alone can deliver conflicting results.sResultsWe successfully established a combined RT-qPCR and S-gene pyrosequencing method which can be optionally exploited after routine diagnostics or for epidemiologic studies. This allows a more reliable interpretation of conflicting RT-qPCR results in specimens with relatively low viral loads and close to the detection limits of qPCR (CT values >30). After laboratory implementation and characterization of a best practice protocol we tested the combined method in a large pediatric cohort from two German medical centers (n=769). Pyrosequencing after RT-qPCR enabled us to uncover 6 previously unrecognized cases of pediatric SARS-CoV-2 associated diseases, partially exhibiting unusual and heterogeneous presentation. Moreover, it is notable that in the course of RT-qPCR/pyrosequencing method establishment we did not observe any case of false-positive diagnosis when confirmed SARS-CoV-2-positive specimens were used from foregoing routine testing.ConclusionsThe proposed protocol allows a specific and sensitive detection of SARS-CoV-2 close to the detection limits of RT-qPCR. Combined RT-qPCR/pyrosequencing does not negatively affect preceding RT-qPCR pipeline in SARS-CoV-2 diagnostics and can be optionally applied in routine to inspect conflicting RT-qPCR results.

For many years it is known that nucleic acids containing runs of guanines can fold into four-stranded structures, G-quadruplexes, but often they were regarded as structural curiosity. Only recently evidence accumulated that these structures occur in vivo and may provide a nucleic-acid-based mechanism for the regulation of important biological processes. Here we review recent progress in understanding the biological function of G-quadruplex structures and the regulation of their formation and resolution in vivo.

To study the role of α4β7 integrin for gut homing of monocytes and to explore the biological consequences of therapeutic α4β7 inhibition with regard to intestinal wound healing.We studied the expression of homing markers on monocyte subsets in the peripheral blood and on macrophage subsets in the gut of patients with IBD and controls with flow cytometry and immunohistochemistry. Integrin function was addressed with dynamic adhesion assays and in vivo gut homing assays. In vivo wound healing was studied in mice deficient for or depleted of α4β7 integrin.Classical and non-classical monocytes were clearly dichotomous regarding homing marker expression including relevant expression of α4β7 integrin on human and mouse non-classical monocytes but not on classical monocytes. Monocyte-expressed α4β7 integrin was functionally important for dynamic adhesion to mucosal vascular addressin cell adhesion molecule 1 and in vivo gut homing. Impaired α4β7-dependent gut homing was associated with reduced (effect size about 20%) and delayed wound healing and suppressed perilesional presence of wound healing macrophages. Non-classical monocytes in the peripheral blood were increased in patients with IBD under clinical treatment with vedolizumab.In addition to reported effects on lymphocytes, anti-α4β7 therapy in IBD also targets non-classical monocytes. Impaired gut homing of such monocytes might lead to a reduction of wound healing macrophages and could potentially explain increased rates of postoperative complications in vedolizumab-treated patients, which have been observed in some studies.

Background In hepatocyte nuclei, hepatitis B virus (HBV) genomes occur episomally as covalently closed circular DNA (cccDNA). The HBV X protein (HBx) is required to initiate and maintain HBV replication. The functional nuclear localization of cccDNA and HBx remains unexplored. Results To identify virus–host genome interactions and the underlying nuclear landscape for the first time, we combined circular chromosome conformation capture (4C) with RNA-seq and ChIP-seq. Moreover, we studied HBx-binding to HBV episomes. In HBV-positive HepaRG hepatocytes, we observed preferential association of HBV episomes and HBx with actively transcribed nuclear domains on the host genome correlating in size with constrained topological units of chromatin. Interestingly, HBx alone occupied transcribed chromatin domains. Silencing of native HBx caused reduced episomal HBV stability. Conclusions As part of the HBV episome, HBx might stabilize HBV episomal nuclear localization. Our observations may contribute to the understanding of long-term episomal stability and the facilitation of viral persistence. The exact mechanism by which HBx contributes to HBV nuclear persistence warrants further investigations. Electronic supplementary material The online version of this article (10.1186/s13072-018-0204-2) contains supplementary material, which is available to authorized users.

Background During sexual reproduction in the unicellular ciliate Stylonychia somatic macronuclei differentiate from germline micronuclei. Thereby, programmed sequence reduction takes place, leading to the elimination of > 95% of germline sequences, which priorly adopt heterochromatin structure via H3K27me3. Simultaneously, 27nt-ncRNAs become synthesized from parental transcripts and are bound by the Argonaute protein PIWI1. Results These 27nt-ncRNAs cover sequences destined to the developing macronucleus and are thought to protect them from degradation. We provide evidence and propose that RNA/DNA base-pairing guides PIWI1/27nt-RNA complexes to complementary macronucleus-destined DNA target sequences, hence transiently causing locally stalled replication during polytene chromosome formation. This spatiotemporal delay enables the selective deposition of temporarily available histone H3.4K27me3 nucleosomes at all other sequences being continuously replicated, thus dictating their prospective heterochromatin structure before becoming developmentally eliminated. Concomitantly, 27nt-RNA-covered sites remain protected. Conclusions We introduce the concept of ‘RNA-induced DNA replication interference’ and explain how the parental functional genome partition could become transmitted to the progeny. Electronic supplementary material The online version of this article (10.1186/s13072-018-0201-5) contains supplementary material, which is available to authorized users.

Background Early pulmonary oxygen exposure is one of the most important factors implicated in the development of bronchopulmonary dysplasia (BPD). Methods Here, we analyzed short- and long-term effects of neonatal hyperoxia on NOS3 and STAT3 expression and corresponding epigenetic signatures using a hyperoxia-based mouse model of BPD. Results Early hyperoxia exposure led to a significant increase in NOS3 (median fold change × 2.37, IQR 1.54–3.68) and STAT3 (median fold change × 2.83, IQR 2.21–3.88) mRNA levels in pulmonary endothelial cells with corresponding changes in histone modification patterns such as H2aZac and H3K9ac hyperacetylation at the respective gene loci. No complete restoration in histone signatures at these loci was observed, and responsivity to later hyperoxia was altered in mouse lungs. In vitro, histone signatures in human aortic endothelial cells (HAEC) remained altered for several weeks after an initial long-term exposure to trichostatin A. This was associated with a substantial increase in baseline eNOS (median 27.2, IQR 22.3–35.6) and STAT3α (median 5.8, IQR 4.8–7.3) mRNA levels with a subsequent significant reduction in eNOS expression upon exposure to hypoxia. Conclusions Early hyperoxia induced permanent changes in histones signatures at the NOS3 and STAT3 gene locus might partly explain the altered vascular response patterns in children with BPD.

Small and long noncoding RNAs (ncRNAs) are key regulators of gene expression. Variations in ncRNA expression patterns can consequently affect the control of many cellular processes. Not just since 2006, when Andrew Z Fire and Craig C Mello were jointly awarded The Nobel Prize in Physiology or Medicine for their discovery of RNA interference, great efforts were undertaken to unleash the biomedical applicability of small noncoding RNAs, in particular microRNAs. With the technological evolution of massive parallel sequencing technologies over the last years, which now are available for an increasing number of scientists, there is a demand for comprehensible and efficient workflows reliable even for unique and valuable clinical specimens. Here we describe a highly reproducible low-cost protocol for analyses of miRNA expression patterns using tagged cDNA libraries and a multiplex sequencing strategy following an Illumina-like protocol. This protocol easily allows the identification of expression differences from samples of tissues of 1-2 mm

Stylonychia lemnae is a classical model single-celled eukaryote, and a quintessential ciliate typified by dimorphic nuclei: A small, germline micronucleus and a massive, vegetative macronucleus. The genome within Stylonychia's macronucleus has a very unusual architecture, comprised variably and highly amplified "nanochromosomes," each usually encoding a single gene with a minimal amount of surrounding noncoding DNA. As only a tiny fraction of the Stylonychia genes has been sequenced, and to promote research using this organism, we sequenced its macronuclear genome. We report the analysis of the 50.2-Mb draft S. lemnae macronuclear genome assembly, containing in excess of 16,000 complete nanochromosomes, assembled as less than 20,000 contigs. We found considerable conservation of fundamental genomic properties between S. lemnae and its close relative, Oxytricha trifallax, including nanochromosomal gene synteny, alternative fragmentation, and copy number. Protein domain searches in Stylonychia revealed two new telomere-binding protein homologs and the presence of linker histones. Among the diverse histone variants of S. lemnae and O. trifallax, we found divergent, coexpressed variants corresponding to four of the five core nucleosomal proteins (H1.2, H2A.6, H2B.4, and H3.7) suggesting that these ciliates may possess specialized nucleosomes involved in genome processing during nuclear differentiation. The assembly of the S. lemnae macronuclear genome demonstrates that largely complete, well-assembled highly fragmented genomes of similar size and complexity may be produced from one library and lane of Illumina HiSeq 2000 shotgun sequencing. The provision of the S. lemnae macronuclear genome sets the stage for future detailed experimental studies of chromatin-mediated, RNA-guided developmental genome rearrangements.

Small and long noncoding RNAs (ncRNAs) are key regulators of gene expression. Variations in ncRNA expression patterns can consequently affect the control of many cellular processes. Not just since 2006, when Andrew Z Fire and Craig C Mello were jointly awarded The Nobel Prize in Physiology or Medicine for their discovery of RNA interference, great efforts were undertaken to unleash the biomedical applicability of small noncoding RNAs, in particular microRNAs. With the technological evolution of massive parallel sequencing technologies over the last years, which now are available for an increasing number of scientists, there is a demand for comprehensible and efficient workflows reliable even for unique and valuable clinical specimens. Here we describe a highly reproducible low-cost protocol for analyses of miRNA expression patterns using tagged cDNA libraries and a multiplex sequencing strategy following an Illumina-like protocol. This protocol easily allows the identification of expression differences from samples of tissues of 1-2 mm3 and fluids of 50-200 μL. We further provide entry points into useful computational biology applications, whose target groups explicitly involve non-bioinformaticians.

Worldwide, chronic hepatitis B virus (HBV) infection is a major health problem and no cure exists. Importantly, hepatocyte intrusion by HBV particles results in a complex deregulation of both viral and host cellular genetic and epigenetic processes. Among the attempts to develop novel therapeutic approaches against HBV infection, several options targeting the epigenomic regulation of HBV replication are gaining attention. These include the experimental treatment with ‘epidrugs’. Moreover, as a targeted approach, the principle of ‘epigenetic editing’ recently is being exploited to control viral replication. Silencing of HBV by specific rewriting of epigenetic marks might diminish viral replication, viremia, and infectivity, even- tually controlling the disease and its complications. Additionally, epigenetic editing can be used as an experimental tool to increase our limited understanding regarding the role of epigenetic modifications in viral infections. Aiming for permanent epigenetic reprogramming of the viral genome without unspecific side effects, this breakthrough may pave the roads for an ambitious technological pursuit: to start designing a curative approach utilizing manipulative molecular therapies for viral infections in vivo. COL0024159

It is well established that G-quadruplex DNA structures form at ciliate telomeres and their formation throughout the cell-cycle by telomere-end-binding proteins (TEBPs) has been analyzed. During replication telomeric G-quadruplex structure has to be resolved to allow telomere replication by telomerase. It was shown that both phosphorylation of TEBPβ and binding of telomerase are prerequisites for this process, but probably not sufficient to unfold G-quadruplex structure in timely manner to allow replication to proceed. Here we describe a RecQ-like helicase required for unfolding of G-quadruplex structures in vivo. This helicase is highly reminiscent of human RecQ protein-like 4 helicase as well as other RecQ-like helicase found in various eukaryotes and E. coli. In situ analyses combined with specific silencing of either the telomerase or the helicase by RNAi and co-immunoprecipitation experiments demonstrate that this helicase is associated with telomerase during replication and becomes recruited to telomeres by this enzyme. In vitro assays showed that a nuclear extract prepared from cells in S-phase containing both the telomerase as well as the helicase resolves telomeric G-quadruplex structure. This finding can be incorporated into a mechanistic model about the replication of telomeric G-quadruplex structures during the cell cycle., Highlights ► Stylonychia StyRecQL is a helicase reminiscent of human RecQ protein-like 4. ► StyRecQL is enriched in replication band of the macronucleus during S-phase. ► StyRecQL colocalizes and is associated with Tert during telomere replication.

Background: Reduced alpha-defensin expression has been reported in the terminal ileum (TI) of adult patients with ileal Crohn's disease (CD). However, little is known about alpha-defensin expression in children with chronic inflammatory bowel disease (IBD). Methods: In all, 283 intestinal biopsies were obtained from children with CD, ulcerative colitis (UC), and healthy controls. Absolute mRNA copy numbers for HD5, HD6, IL-8, Villin 1, and Tcf-4 were analyzed by reverse-transcription polymerase chain reaction (RT-PCR). HD5 immunostaining was performed on biopsy sections and patients genotyped for NOD2 mutations. Results: Equal expression levels of alpha-defensins (HD5 and HD6) were found in TI biopsies of children with ileal CD (L1+L3) compared to patients with colonic disease (L2) and healthy controls. In contrast, we found significantly higher levels of alpha-defensins in the TI of children with UC compared to CD and controls. Reduced expression of Tcf-4 was observed exclusively in the duodenum and TI of CD patients with L1+L3 phenotype. We demonstrate significantly increased expression of HD5 and HD6 in the inflamed colon of IBD children (UC and CD) attributable to the presence of metaplastic Paneth cells. Conclusions: In this study no difference in alpha-defensin expression was found in the TI of CD children and controls. However, significant reduction of Tcf-4 in L1+L3 phenotype suggests that a possibly impaired PC differentiation may lead to altered HD5 and HD6 expression at some stage of disease. Additionally, substantially increased expression of alpha-defensins in the inflamed colonic mucosa of children with IBD raises the question for their potential involvement in modulating inflammation in these patients. (Inflamm Bowel Dis 2010;)

De novo addition of telomeric sequences can occur at broken chromosomes and must be well controlled, which is essential during programmed DNA reorganization processes. In ciliated protozoa an extreme form of DNA-reorganization is observed during macronuclear differentiation after sexual reproduction leading to the elimination of specific parts of the germline genome. Regulating these processes involves small noncoding RNAs, but in addition DNA-reordering, excision and amplification require RNA templates deriving from the parental macronucleus. We show that these putative RNA templates can carry telomeric repeats. Microinjection of RNA templates carrying modified telomeres into the developing macronucleus leads to modified telomeres in vegetative cells, providing strong evidence, that de novo addition of telomeres depends on a telomere-containing transcript from the parental macronucleus.

Background and objective: The study determined growth factor gene expression obtained from patients undergoing curative total mesorectal excision (TME) between 2009 and 2016 at the Helios Klinikum Wuppertal; Germany (total of n=44). Methods: Rectal cancers were grouped according to Lauren’s classification (UICC I-IV stages); overall survival (OS) and progression free survival (PFS). 25 patients were in UICC III/IV stages, while 19 patients were in UICC I/II stages. VEGF-A/B/C/D, PDGF-A, EGF, IGF-1/2, and corresponding receptor subtypes 1/2 mRNA enrichment was determined using quantitative real-time PCR. Data were correlated to clinical outcome. Results: The obtained results show that VEGF-B and IGF-2 mRNA were significantly higher expressed in rectal adenocarcinoma than adjacent normal tissues, while other GF genes were not significantly affected. IGF-2 levels were further upregulated in advanced stages UICC III/IV where patients received radiotherapy. Patients with good response to neoadjuvant radiotherapy (as determined by the histological Dworak regression score) had significant better overall survival. Expression of microRNA miR-483 (a known microRNA molecule previously associated with IGF-2 expression) was evaluated by TaqMAN PCR, but no significant correlation to IGF-2 levels was detected. Conclusions: IGF-2 appears as a biomarker of rectal adenocarcinoma, but does not serve as predictive parameter for tumor progression or survival. The results encourage further development of biomarkers targeting IGF-2 molecules in blood or urine. Furthermore, IGF-2 levels are increased in patients undergoing radiotherapy and thus, IGF related molecules might also serve as predictors for response to neoadjuvant therapy in rectal adenocarcinoma.

Epigenetics can be defined as stable, potentially heritable changes in the cellular phenotype caused by mechanisms other than alterations to the underlying DNA sequence. As such, any observed phenotypic changes including organ development, aging, and the occurrence of disease could be driven by epigenetic mechanisms in the presence of stable cellular DNA sequences. Indeed, with the exception of rare mutations, the human genome-sequence has remained remarkably stable over the past centuries. In contrast, substantial changes to our environment as part of our modern life style have not only led to a significant reduction of certain infectious diseases but also seen the exponential increase in complex traits including obesity and multifactorial diseases such as autoimmune disorders. It is becoming increasingly clear that epigenetic mechanisms operate at the interface between the genetic code and our environment, and a large body of existing evidence supports the importance of environmental factors such as diet and nutrition, infections, and exposure to toxins on human health. This seems to be particularly the case during vulnerable periods of human development such as pregnancy and early life. Importantly, as the first point of contact for many of such environmental factors including nutrition, the digestive system is being increasingly linked to a number of "modern" pathologies. In this review article, we aim to give a brief introduction to the basic molecular principals of epigenetics and provide a concise summary of the existing evidence for the role of epigenetic mechanisms in gastrointestinal health and disease, hepatology, and nutrition.

Aim Whereas enteral nutrition has been recognized to play an important role for the development of necrotizing enterocolitis (NEC), the exact pathomechanism is still under debate [1,2]. In this study we aimed to analyze how introduction of enteral foods affects intestinal gene regulation and chromatin structure of different inflammatory and pattern recognition genes (i.e. IL8, TLR2, TLR4, REG3A) in premature pigs before any clinical symptoms of NEC.

Aim: To analyze how enteral food introduction affects intestinal gene regulation and chromatin structure in preterm pigs. Materials & methods: Preterm pigs were fed parenteral nutrition plus/minus slowly increasing volumes of enteral nutrition. Intestinal gene-expression and chromatin structure were analyzed 5 days after birth. Results: Enteral feeding led to differential upregulation of inflammatory and pattern recognition receptor genes, including IL8 (median: 5.8, 95% CI: 3.9–7.8 for formula; median: 2.2, 95% CI: 1.3–3.3 for colostrum) and TLR4 (median: 3.7, 95% CI: 2.6–4.8 for formula; no significant differences for colostrum) with corresponding decondensed chromatin configurations. On histology this correlated with mild mucosal lesions, particularly in formula-fed pigs. In CaCo-2 cells, histone hyperacetylation led to a marked increase in TLR4 mRNA and increased IL8 expression upon stimulation with lipopolysaccharide (median: 7.0; interquartile range: 5.63–8.85) compared with naive cells (median 4.2; interquartile range: 2.45–6.33; p = 0.03). Conclusion: Enteral feeding, particular with formula, induces subclinical inflammation in the premature intestine and more open chromatin structure in key inflammatory genes. This may increase the susceptibility for necrotizing enterocolitis.

Several models for specific excision of micronucleus-specific DNA sequences during macronuclear development in ciliates exist. While the template-guided recombination model suggests recombination events resulting in specific DNA excision and reordering of macronucleus-destined sequences (MDS) guided by a template, there is evidence that an RNA interference-related mechanism is involved in DNA elimination in holotrichous ciliates. We describe that in the stichotrichous ciliate Stylonychia , snRNAs homologous to micronucleus-specific sequences are synthesized during macronuclear differentiation. Western and in situ analyses demonstrate that histone H3 becomes methylated at K9 de novo during macronuclear differentiation, and chromatin immunoprecipitation revealed that micronucleus-specific sequences are associated with methylated H3. To link both observations, expression of a PIWI homolog, member of the RNA-induced silencing complex, was silenced. In these cells, the methylated micronucleus-specific histone H3 variant “X” is still present in macronuclear anlagen and no K9 methylation of histone H3 is observed. We suggest that snRNA recruits chromatin-modifying enzymes to sequences to be excised. Based on our and earlier observations, we believe that this mechanism is not sufficient for specific excision of sequences and reordering of MDS in the developing macronucleus and propose a model for internal eliminated sequence excision and MDS reordering in stichotrichous ciliates.

Telomere end-binding proteins (TEBPs) bind to the guanine-rich overhang (G-overhang) of telomeres. Although the DNA binding properties of TEBPs have been investigated in vitro, little is known about their functions in vivo. Here we use RNA interference to explore in vivo functions of two ciliate TEBPs, TEBPalpha and TEBPbeta. Silencing the expression of genes encoding both TEBPs shows that they cooperate to control the formation of an antiparallel guanine quadruplex (G-quadruplex) DNA structure at telomeres in vivo. This function seems to depend on the role of TEBPalpha in attaching telomeres in the nucleus and in recruiting TEBPbeta to these sites. In vitro DNA binding and footprinting studies confirm the in vivo observations and highlight the role of the C terminus of TEBPbeta in G-quadruplex formation. We have also found that G-quadruplex formation in vivo is regulated by the cell cycle-dependent phosphorylation of TEBPbeta.

Spatial and temporal replication patterns are used to describe higher-order chromatin organisation from nuclei of early metazoan to mammalian cells. Here we demonstrate evolutionary conserved similarities and differences in replication patterns of micronuclei and macronuclei in the spirotrichous ciliate Stylonychia lemnae. Since this organism possesses two kinds of morphologically and functionally different nuclei in one cell, it provides an excellent model system to analyse topological requirements for DNA replication and transcription.Replication in the heterochromatic micronucleus occurs in foci-like structures showing spatial and temporal patterns similar to nuclei of higher eukaryotes, demonstrating that these patterns are inherent features of nuclear architecture. The `nanochromosomes' of the macronucleus are replicated in the propagating replication band. We show that it consists of hundreds of replication foci. Post-replicative macronuclear chromatin remains organised in foci. These foci are not randomly distributed throughout the macronucleus, indicating a higher-order organisation of macronuclear chromatin above the level of `nanochromosomes'. Both telomerase and proliferating cell nuclear antigen (PCNA) occur as foci-like structures in the rear zone of the replication band, suggesting that a wave of chromatin modification driven by a short or continuous exogenous signal permits the assembly of replication factories at predicted sites. We further show that transcription occurs at discrete sites colocalised with putative nucleoli and dispersed chromatin.Common principles of functional nuclear architecture were conserved during eukaryotic evolution. Moreover nuclear duality inherent to ciliates with their germline micronucleus and their somatic macronucleus may eventually provide further insight into epigenetic regulation of transcription, replication and nuclear differentiation.

Telomerase is a specialized reverse transcriptase synthesizing DNA repeats at telomeres. In addition to the RNA and catalytic protein components, telomerase from the ciliate Euplotes aediculatus contains the subunit p43. This protein is homologous to the La autoantigen, functioning in maturation of RNA polymerase III transcripts. Here we provide evidence that p43 is primarily associated with the telomerase ribonucleoprotein in vivo. Recombinant p43 binds telomerase RNA with low-nanomolar affinity in vitro, recognizing stem I and adjacent nucleotides or structures in the core of the RNA. Unlike authentic La proteins, p43 does not bind strongly to RNA polymerase III precursor transcripts and does not exhibit a marked binding preference for 3'-terminal oligouridylate residues. In isolated macronuclei, p43 largely colocalizes with telomerase RNA in discrete foci. These findings suggest that p43 is not the Euplotes La protein but instead plays a dedicated role in telomerase assembly and/or function. Thus, p43 joins the telomerase reverse transcriptase and the yeast proteins Est1p and Est3p as the only telomerase-specific proteins identified so far.

The use of small double-stranded RNA to inhibit expression of genes has proven to be a powerful tool to analyze the biological function of genes in various eukaryotic organisms. We have established the RNAi technology in three genera of spirotrichous ciliates, Euplotes, Oxytricha and Stylonychia . Here we describe this method to silence gene expression in these three genera. Expression of the following genes was inhibited: the catalytic subunit of telomerase (TERT) and the telomerase-associated protein p43 in Euplotes and the a-subunit of the telomere-binding protein (αTeBP) of Stylonychia and Oxytricha . In addition, we report how the biological function of a developmentally regulated gene can be elucidated by this technique in Stylonychia.

Macronuclear DNA of stichotrichous ciliates occurs in small ‘gene-sized’ molecules with sizes of about 0.5 to 40 kb. Each of these molecules is terminated by telomeric sequences of defined length. A single macronucleus contains up to 108 DNA molecules; due to the high concentration of telomeric sequences in this nucleus it is an attractive model to study telomere behaviour. We recently provided evidence that macronuclear telomeres are attached to the nuclear matrix and that this interaction is mediated by the telomere binding protein (TeBP). Using various experimental approaches, we now demonstrate that telomeres as well as both subunits of the telomere binding protein are associated with the nuclear matrix. However, there is no direct binding of telomeric DNA to the matrix but telomere matrix interaction is exclusively mediated by the TeBP. In addition, we show that telomeric sequences adopt in vivo the antiparallel G-quartet structure when bound to the nuclear matrix. These data not only allow us to propose a model for macronuclear architecture but may also be relevant for further analysis of telomere–matrix interactions in higher eukaryotes.

Results In human fetuses NOS3 expression directly correlated with 5-hydroxymethylcytosine levels as well as H3K9ac and H2A.Zac at the transcription start site. Using an in vitro model for placental dysfunction we confirmed the dynamic NOS3 response and histone acetylation patterns. Concomitantly we recognized massive turnover of Stat3 at a discrete binding site in the NOS3 promoter upon hypoxic conditions. Moreover, knock-down experiments targeting either STAT3 or NOS3 provided strong evidence for a functional NOS3 and STAT3 relationship. Interestingly, induced hyperacetylation and in vitro reporter assays showed that NOS3 expression becomes self-attenuated by co-expression of intronic 27nt-ncRNA modulating STAT3 signaling.

Background Virus-host interactions result in altered gene expression profiles in host cell nuclei and enable virus particle production, thus obligatorily involving changes in their epigenomes. Neither such epigenome changes nor their response to antiviral treatment have been extensively studied to date, although viral infections are known to contribute to the long-term development of severe secondary diseases, for example, hepatocellular carcinoma. This may be causally linked to virus-induced persistent plastic chromatin deformations. Results We studied whether impaired hepatitis B virus (HBV) replication can lead to the restitution of epigenome signatures hypothesizing that hepatocytes alternatively could adopt a ‘memory’ state of the infection; that is, the chromatin could persist in a HBV-induced configuration potentially inheritable between dividing hepatocytes. We therefore determined epigenomic signatures and gene expression changes altered by HBV and the effects of suppressed HBV replication in nontransformed hepatocytes of newborn mice. Further we investigated differential histone acetyltransferase and histone deacetylase activities in HBV-negative and HBVpositive hepatocytes, as well as the effects of HBV suppression on gene expression and the chromatin landscape. We show that the expression of several genes and the chromatin landscape become altered upon HBV infection, including global hypoacetylation of H2A.Z and H3K9. Reporter assays monitoring the activities of histone acetyltransferases or histone deacetylases, respectively, suggest that hypoacetylation most probably depends on elevated sirtuin deacetylase activity, but not on class I/II histone deacetylases. Using Micrococcus nuclease to study the chromatin accessibility in met murine-D3 and hepatitis B virus met murine hepatocytes, we demonstrate that the observed differences in H2A.Z/H3K9 acetylation lead to global chromatin structure changes. At all selected sites examined by chromatin immunoprecipitation and quantitative real-time PCR, these effects can be partly restituted via the nucleoside analog reverse transcriptase inhibitor 3TC or using anti-HBV microRNA-like molecules. Conclusions Increased sirtuin activity might lead to global histone hypoacetylation signatures, which could contribute to the HBV-induced pathomechanism in nontransformed hepatocytes. Using several techniques to suppress HBV replication, we observed restituted gene expression and chromatin signature patterns reminiscent of noninfected hepatocytes. Importantly, ectopic expression of antiviral short-hairpin RNA, but not microRNA-like molecules, provoked intolerable off-target effects on the gene expression level. Electronic supplementary material The online version of this article (doi:10.1186/1868-7083-6-26) contains supplementary material, which is available to authorized users.

Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3′-terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T 4 G 4 ), forms a 16-nucleotide 3′-overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro . Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structure in vivo has been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by the Stylonychia telomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity of K d = 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar ( K d = 3–5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus of Stylonychia lemnae . The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres of Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.

Background Regulation of chromatin structure involves deposition of selective histone variants into nucleosome arrays. Numerous histone H3 variants become differentially expressed by individual nanochromosomes in the course of macronuclear differentiation in the spirotrichous ciliate Stylonychia lemnae. Their biological relevance remains to be elucidated. Results We show that the differential assembly of H3 variants into chromatin is strongly correlated with the functional separation of chromatin structures in developing macronuclei during sexual reproduction in Stylonychia, thus probably determining the fate of specific sequences. Specific H3 variants approximately 15 kDa or 20 kDa in length are selectively targeted by post-translational modifications. We found that only the 15 kDa H3 variants including H3.3 and H3.5, accumulate in the early developing macronucleus, and these also occur in mature macronuclei. H3.7 is a 20 kDa variant that specifically becomes enriched in macronuclear anlagen during chromosome polytenization. H3.7, acetylated at lysine-32 (probably equivalent to lysine-36 of most H3 variants), is specifically associated with a sequence class that is retained in the mature macronucleus and therefore does not undergo developmental DNA elimination. H3.8 is another 20 kDa variant that is restricted to the micronucleus. H3.8 is selectively targeted by lysine methylation and by serine or threonine phosphorylation. Intriguingly, the expression and chromatin localization of the histone variant H3.3 was impaired during macronuclear differentiation after RNA interference knock-down of Piwi expression. Conclusions Differential deposition of H3 variants into chromatin strongly correlates with the functional distinction of genomic sequence classes on the chromatin level, thus helping to determine the fate of specific DNA sequences during sexual reproduction in Stylonychia. Consequently, H3 variants are selectively targeted by post-translational modifications, possibly as a result of deviations within the recognition motifs, which allow binding of effector proteins. We propose that differential assembly of histone variants into chromatin of various nuclear types could contribute to nuclear identity, for example, during differential development of either new micronuclei or a macronuclear anlage from mitosis products of the zygote nucleus (synkaryon). The observation that the Piwi-non-coding RNA (ncRNA) pathway influences the expression and deposition of H3.3 in macronuclear anlagen indicates for the first time that selective histone variant assembly into chromatin might possibly depend on ncRNA.

BACKGROUND AND OBJECTIVES Genotype changes have been observed during long-term follow-up in adults with chronic hepatitis B virus (HBV) infection. The aim of this study was to assess the frequency and distribution of HBV genotype shifts in treated and untreated European children during the different phases of chronic hepatitis B. METHODS The initial population consisted of 85 HBeAg-positive children who were sequentially investigated for HBV genotypes for a mean period of 7.1 years. During the survey, 65 patients seroconverted to anti-HBe and 24 received antiviral treatment. Genotyping was done by restriction fragment length polymorphism. RESULTS Genotypes at enrollment were distributed as follows: D (n = 67), A (n = 11), B (n = 4), and C and mixed (n = 3). A genotype change was observed in 16 (19%) participants at last visit, of whom 1 of 20 (5%) remained persistently HBeAg positive and 15 of 65 (23%) children had seroconverted to anti-HBe (P < 0.001). The genotype shift was D to A or B in 9 individuals, A to D in 5, and A to B in 1. Genotype changes were more frequent in untreated than in treated subjects; however, the difference was not statistically significant. CONCLUSIONS The results of this study suggest that genotype change is a relatively frequent event in children with chronic hepatitis B after anti-HBe seroconversion and independent of treatment. The reasons why only some patients will experience this event remain to be clarified.

Aim of the study To contribute to our understanding of DNA and chromatin-modifying mechanisms modulated by HBV, which are responsible for the transregulation of host cells for virus particle production and malignant transformation of hepatocytes. We further study the effect of lamivudine treatment or an antiviral vector-based RNAi strategy targeting HBx on the establishment and maintenance of DNA methylation patterns. Our research not only aims to uncover epigenomic plasticity modulated by HBV. We also intend to contribute to the open problem, whether therapies lead to the restoration of regular epigenomic signatures, or whether hepatocytes persist in a deregulated epigenetic ‘memory’ state of HBV infection thus still carrying the risk for malignant transformation.

Objective. To describe the development of the gut microbiota in extremely low birth weight (ELBW) infants with and without necrotizing enterocolitis (NEC) between April 2008 and December 2009, fecal microflora was prospectively analyzed in fecal samples of all ELBW infants using real-time PCR assays. In addition, fecal inflammatory were measured.Results. Fecal microflora established early in ELBW infants and microbiota composition remained stable over the first 28 days of life except for the prevalence ofC. difficilewhich decreased with decreasing antibiotic use. Infants who subsequently developed NEC had an increase of total bacterial count (9.8-fold) 24 h prior to clinical symptoms mainly due to the expansion ofE. colispecies (21.6-fold), whereas microbiota composition did not differ from healthy ELBW infants five days before onset of NEC. Importantly, S100A12 and hBD2 positively correlated with the total andE. colibacterial CFU/g feces (r20.4 and 0.64, resp.).Conclusions. In summary, we found evidence for a disturbed homeostasis between the intestinal microbiome and host immunity in ELBW infants with NEC. Moreover, S100A12 and hBD2 correlate with the fecal microbiota thus linking the intestinal innate immune response to the bacterial colonization thus possibly providing a diagnostic tool in the future.

The aim of this study was to analyze whether the mucosal innate immune response of extremely-low-birth-weight (ELBW) infants might play a role in the development of necrotizing enterocolitis (NEC). Between April 2008 and December 2009 antimicrobial peptides were prospectively measured in fecal samples of ELBW infants. In cases requiring abdominal surgery, full-thickness gut biopsies were analyzed for expression of human β-defensin 2 (hBD2), interleukin-8 (IL-8), villin, MD2, and Toll-like receptor 4 (TLR4). Fecal hBD1 concentrations were consistently low in all patients, whereas hBD2 concentrations were high in meconium, particularly in clinical chorioamnionitis, and then dropped, followed by a steady increase after day 14. Infants with moderate NEC showed significantly increased fecal hBD2 concentrations before clinical symptoms, in contrast to infants developing severe NEC. Analysis of intestinal resection material obtained from patients with severe NEC revealed low hBD2 mRNA and protein levels, and increased expression of the inflammatory cytokine IL-8. High hBD2 concentrations, reflecting strong intestinal immune responses, were associated with moderate courses of the disease. In severe NEC, low hBD2 expression was accompanied by low TLR4/MD2 expression, suggesting an inadequate response to luminal bacteria, possibly predisposing those infants to the development of NEC.