Uncovering the gastrointestinal passage, intestinal epithelial cellular uptake, and AGO2 loading of milk miRNAs in neonates using xenomiRs as tracers.

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.

An Adenoviral Vector as a Versatile Tool for Delivery and Expression of miRNAs.

Only two decades after discovering miRNAs, our understanding of the functional effects of deregulated miRNAs in the development of diseases, particularly cancer, has been rapidly evolving. These observations and functional studies provide the basis for developing miRNA-based diagnostic markers or new therapeutic strategies. Adenoviral (Ad) vectors belong to the most frequently used vector types in gene therapy and are suitable for strong short-term transgene expression in a variety of cells. Here, we report the set-up and functionality of an Ad-based miRNA vector platform that can be employed to deliver and express a high level of miRNAs efficiently. This vector platform allows fast and efficient vector production to high titers and the expression of pri-miRNA precursors under the control of a polymerase II promoter. In contrast to non-viral miRNA delivery systems, this Ad-based miRNA vector platform allows accurate dosing of the delivered miRNAs. Using a two-vector model, we showed that Ad-driven miRNA expression was sufficient in down-regulating the expression of an overexpressed and highly stable protein. Additional data corroborated the downregulation of multiple endogenous target RNAs using the system presented here. Additionally, we report some unanticipated synergistic effects on the transduction efficiencies in vitro when cells were consecutively transduced with two different Ad-vectors. This effect might be taken into consideration for protocols using two or more different Ad vectors simultaneously.

Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology.

The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Among available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, as it is mobile, scalable, and cost-effective. Therefore, streamlined nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification. We adapted and simplified existing workflows using the 'midnight' 1200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost the entire SARS-CoV-2 genome. Subsequently, we applied Oxford Nanopore Rapid Barcoding and the portable MinION Mk1C sequencer combined with the interARTIC bioinformatics pipeline. We tested a simplified and less time-consuming workflow using SARS-CoV-2-positive specimens from clinical routine and identified the CT value as a useful pre-analytical parameter, which may help to decrease sequencing failures rates. Complete pipeline duration was approx. 7 h for one specimen and approx. 11 h for 12 multiplexed barcoded specimens. The adapted protocol contains fewer processing steps and can be completely conducted within one working day. Diagnostic CT values deduced from qPCR standardization experiments can act as principal criteria for specimen selection. As a guideline, SARS-CoV-2 genome copy numbers lower than 4 × 106 were associated with a coverage threshold below 20-fold and incompletely assembled SARS-CoV-2 genomes. Thus, based on the described thermocycler/chemistry combination, we recommend CT values of ~26 or lower to achieve full and high-quality SARS-CoV-2 (+)RNA genome coverage.

Combined RT-qPCR and pyrosequencing of a Spike glycoprotein polybasic cleavage motif can uncover pediatric SARS-CoV-2 infections associated with heterogeneous presentation.

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.

Biogenesis of Developmental Master Regulatory 27nt-RNAs in Stylonychia-Can Coding RNA Turn into Non-Coding?

In the ciliate Stylonychia, somatic macronuclei differentiate from germline micronuclei during sexual reproduction, accompanied by developmental sequence reduction. Concomitantly, over 95% of micronuclear sequences adopt a heterochromatin structure characterized by the histone variant H3.4 and H3K27me3. RNAi-related genes and histone variants dominate the list of developmentally expressed genes. Simultaneously, 27nt-ncRNAs that match sequences retained in new macronuclei are synthesized and bound by PIWI1. Recently, we proposed a mechanistic model for 'RNA-induced DNA replication interference' (RIRI): during polytene chromosome formation PIWI1/27nt-RNA-complexes target macronucleus-destined sequences (MDS) by base-pairing and temporarily cause locally stalled replication. At polytene chromosomal segments with ongoing replication, H3.4K27me3-nucleosomes become selectively deposited, thus dictating the prospective heterochromatin structure of these areas. Consequently, these micronucleus-specific sequences become degraded, whereas 27nt-RNA-covered sites remain protected. However, the biogenesis of the 27nt-RNAs remains unclear. It was proposed earlier that in stichotrichous ciliates 27nt-RNA precursors could derive from telomere-primed bidirectional transcription of nanochromosomes and subsequent Dicer-like (DCL) activity. As a minimalistic explanation, we propose here that the 27nt-RNA precursor could rather be mRNA or pre-mRNA and that the transition of coding RNA from parental macronuclei to non-coding RNAs, which act in premature developing macronuclei, could involve RNA-dependent RNA polymerase (RDRP) activity creating dsRNA intermediates prior to a DCL-dependent pathway. Interestingly, by such mechanism the partition of a parental somatic genome and possibly also the specific nanochromosome copy numbers could be vertically transmitted to the differentiating nuclei of the offspring.

27nt-RNAs guide histone variant deposition via 'RNA-induced DNA replication interference' and thus transmit parental genome partitioning in Stylonychia.

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.

RNA-template dependent de novo telomere addition.

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.

CpG signalling, H2A.Z/H3 acetylation and microRNA-mediated deferred self-attenuation orchestrate foetal NOS3 expression.

BACKGROUND: An adverse intrauterine environment leads to permanent physiological changes including vascular tone regulation, potentially influencing the risk for adult vascular diseases. We therefore aimed to monitor responsive NOS3 expression in human umbilical artery endothelial cells (HUAEC) and to study the underlying epigenetic signatures involved in its regulation. RESULTS: NOS3 and STAT3 mRNA levels were elevated in HUAEC of patients who suffered from placental insufficiency. 5-hydroxymethylcytosine, H3K9ac and Histone 2A (H2A).Zac at the NOS3 transcription start site directly correlated with NOS3 mRNA levels. Concomitantly, we observed entangled histone acetylation patterns and NOS3 response upon hypoxic conditions in vitro. Knock-down of either NOS3 or STAT3 by RNAi provided evidence for a functional NOS3/STAT3 relationship. Moreover, we recognized massive turnover of Stat3 at a discrete binding site in the NOS3 promoter. Interestingly, induced hyperacetylation resulted in short-termed increase of NOS3 mRNA followed by deferred decrease indicating that NOS3 expression could become self-attenuated by co-expressed intronic 27 nt-ncRNA. Reporter assay results and phylogenetic analyses enabled us to propose a novel model for STAT3-3'-UTR targeting by this 27-nt-ncRNA. CONCLUSIONS: An adverse intrauterine environment leads to adaptive changes of NOS3 expression. Apparently, a rapid NOS3 self-limiting response upon ectopic triggers co-exists with longer termed expression changes in response to placental insufficiency involving differential epigenetic signatures. Their persistence might contribute to impaired vascular endothelial response and consequently increase the risk of cardiovascular disease later in life.