SARS-CoV-2 Vaccine Alpha and Delta Variant Breakthrough Infections Are Rare and Mild but Can Happen Relatively Early after Vaccination.

(1) Background: Some COVID-19 vaccine recipients show breakthrough infection. It remains unknown, which factors contribute to risks and severe outcomes. Our aim was to identify risk factors for SCoV2 breakthrough infections in fully vaccinated individuals. (2) Methods: We conducted a retrospective case-control study from 28 December 2020 to 25 October 2021. Data of all patients with breakthrough infection was compared to data of all vaccine recipients in the Canton of Basel-City, Switzerland. Further, breakthrough infections by Alpha- and Delta-variants were compared. (3) Results: Only 0.39% (488/126,586) of all vaccine recipients suffered from a breakthrough infection during the observational period, whereof most cases were asymptomatic or mild (97.2%). Breakthrough infections after full vaccination occurred in the median after 78 days (IQR 47-123.5). Factors with lower odds for breakthrough infection were age (OR 0.987) and previous COVID-19 infection prior to vaccination (OR 0.296). Factors with higher odds for breakthrough infection included vaccination with Pfizer/BioNTech instead of Moderna (OR 1.459), chronic disease (OR 2.109), and healthcare workers (OR 1.404). (4) Conclusions: Breakthrough infections are rare and mild but can occur early after vaccination. This implies that booster vaccination might be initiated earlier, especially for risk groups. Due to new variants emerging repeatedly, continuous monitoring of breakthrough infections is crucial.

Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach: a multi-institutional research study.

BACKGROUND: Gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material. METHODS: We describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples. RESULTS: Reproducibility between laboratories using diluted fusion-positive cell lines was 100%. A cohort of lung clinical research samples from different origins (tissue biopsies, tissue resections, lymph nodes and pleural fluid samples) were used to evaluate the panel. We observed 97% concordance for ALK (28/30 positive; 71/70 negative samples), 95% for ROS1 (3/4 positive; 19/18 negative samples), and 93% for RET (2/1 positive; 13/14 negative samples) between the AmpliSeq assay and other methodologies. CONCLUSION: This methodology enables simultaneous detection of multiple ALK, ROS1, RET, and NTRK1 gene fusion transcripts in a single panel, enhanced by an integrated analysis solution. The assay performs well on limited amounts of input RNA (10 ng) and offers an integrated single assay solution for detection of actionable fusions in lung adenocarcinoma, with potential savings in both cost and turn-around-time compared to the combination of all four assays by other methods.

Development of a semi-conductor sequencing-based panel for genotyping of colon and lung cancer by the Onconetwork consortium.

BACKGROUND: The number of predictive biomarkers that will be necessary to assess in clinical practice will increase with the availability of drugs that target specific molecular alterations. Therefore, diagnostic laboratories are confronted with new challenges: costs, turn-around-time and the amount of material required for testing will increase with the number of tests performed on a sample. Our consortium of European clinical research laboratories set out to test if semi-conductor sequencing provides a solution for these challenges. METHODS: We designed a multiplex PCR targeting 87 hotspot regions in 22 genes that are of clinical interest for lung and/or colorectal cancer. The gene-panel was tested by 7 different labs in their own clinical setting using ion-semiconductor sequencing. RESULTS: We analyzed 155 samples containing 112 previously identified mutations in the KRAS, EGFR en BRAF genes. Only 1 sample failed analysis due to poor quality of the DNA. All other samples were correctly genotyped for the known mutations, even as low as 2%, but also revealed other mutations. Optimization of the primers used in the multiplex PCR resulted in a uniform coverage distribution over the amplicons that allows for efficient pooling of samples in a sequencing run. CONCLUSIONS: We show that a semi-conductor based sequencing approach to stratify colon and lung cancer patients is feasible in a clinical setting.

Analysis of Piwi-loaded small RNAs in Tetrahymena.

Scan RNAs (scnRNAs) are developmentally regulated siRNAs of ~26-32 nucleotides in length that are involved in programmed DNA elimination in Tetrahymena. scnRNAs are loaded onto the Piwi-related protein Twi1p and 2'-O-methylated at their 3' termini. We describe two alternative strategies for analyzing the Twi1p-loaded scnRNAs: preparation of loaded scnRNAs by immuno-purification of the Twi1p-scnRNA complex and exclusion of non-methylated scnRNAs during cDNA library construction using periodate oxidation.

Loading and pre-loading processes generate a distinct siRNA population in Tetrahymena.

The various properties of small RNAs, such as length, terminal nucleotide, thermodynamic asymmetry and duplex mismatches, can impact their sorting into different Argonaute proteins in diverse eukaryotes. The developmentally regulated 26- to 32-nt siRNAs (scnRNAs) are loaded to the Argonaute protein Twi1p and display a strong bias for uracil at the 5' end. In this study, we used deep sequencing to analyze loaded and unloaded populations of scnRNAs. We show that the size of the scnRNA is determined during a pre-loading process, whereas their 5' uracil bias is attributed to both pre-loading and loading processes. We also demonstrate that scnRNAs have a strong bias for adenine at the third base from the 3' terminus, suggesting that most scnRNAs are direct Dicer products. Furthermore, we show that the thermodynamic asymmetry of the scnRNA duplex does not affect the guide and passenger strand decision. Finally, we show that scnRNAs frequently have templated uracil at the last base without a strong bias for adenine at the second base indicating non-sequential production of scnRNAs from substrates. These findings provide a biochemical basis for the varying attributes of scnRNAs, which should help improve our understanding of the production and turnover of scnRNAs in vivo.

Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena.

The ciliated protozoan Tetrahymena undergoes extensive programmed DNA elimination when the germline micronucleus produces the new macronucleus during sexual reproduction. DNA elimination is epigenetically controlled by DNA sequences of the parental macronuclear genome, and this epigenetic regulation is mediated by small RNAs (scan RNAs [scnRNAs]) of ∼28-30 nucleotides that are produced and function by an RNAi-related mechanism. Here, we examine scnRNA production and turnover by deep sequencing. scnRNAs are produced exclusively from the micronucleus and nonhomogeneously from a variety of chromosomal locations. scnRNAs are preferentially derived from the eliminated sequences, and this preference is mainly determined at the level of transcription. Despite this bias, a significant fraction of scnRNAs is also derived from the macronuclear-destined sequences, and these scnRNAs are degraded during the course of sexual reproduction. These results indicate that the pattern of DNA elimination in the new macronucleus is shaped by the biased transcription in the micronucleus and the selective degradation of scnRNAs in the parental macronucleus.

The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus.

Emerging evidence suggests that RNA interference (RNAi)-related processes act both in the cytoplasm and in the nucleus. However, the process by which the RNAi machinery is transported into the nucleus remains poorly understood. The Tetrahymena Argonaute protein Twi1p localizes to the nucleus and is crucial for small RNA-directed programmed DNA elimination. In this study, we identify Giw1p, which binds to Twi1p and is required for its nuclear localization. Furthermore, the endoribonuclease (Slicer) activity of Twi1p plays a vital role in the removal of one of the two strands of Twi1p-associated small interfering RNAs (siRNAs), leading to a functionally mature Twi1p-siRNA complex. Slicer activity is also shown to be required for nuclear localization of Twi1p and for its association with Giw1p. These results suggest that Giw1p senses the state of Twi1p-associated siRNAs and selectively transports the mature Twi1p-siRNA complex into the nucleus.

Non-coding RNA: a bridge between small RNA and DNA.

Small RNAs are not only involved in post-transcriptional gene silencing in the cytoplasm, but also modulate activities of the nuclear genome. Although silencing of mRNAs involves RNA-RNA base-pairing, it remains unclear whether the RNAi machinery responsible for chromatin or DNA modification interacts with DNA or RNA. The identification of an RNA helicase required for chromatin-Argonaute protein interaction as well as the presence of Argonaute-associated long non-coding transcripts in Tetrahymena suggest the presence of RNA-RNA interactions prior to small RNA-induced heterochromatin formation in ciliates.

2'-O-methylation stabilizes Piwi-associated small RNAs and ensures DNA elimination in Tetrahymena.

Small RNAs approximately 20-30 nucleotides (nt) in length regulate gene expression at the transcriptional and post-transcriptional levels. In the plant Arabidopsis, all small RNAs are 3'-terminal 2'-O-methylated by HEN1, whereas only a subset of small RNAs carry this modification in metazoans. This methylation is known to stabilize small RNAs, but its biological significance remains unclear. In the ciliated protozoan Tetrahymena thermophila, two classes of small RNAs have been identified: RNAs approximately 28-29 nt long (scnRNAs) that are expressed only during sexual reproduction, and constitutively expressed approximately 23-24 nt siRNAs. In this study, we demonstrate that scnRNAs, but not siRNAs, are 2'-O-methylated at their 3' ends. The Tetrahymena HEN1 homolog Hen1p is responsible for scnRNA 2'-O-methylation. Loss of Hen1p causes a gradual reduction in the level and length of scnRNAs, defects in programmed DNA elimination, and inefficient production of sexual progeny. Therefore, Hen1p-mediated 2'-O-methylation stabilizes scnRNA and ensures DNA elimination in Tetrahymena. This study clearly shows that 3'-terminal 2'-O-methylation on a selected class of small RNAs regulates the function of a specific RNAi pathway.