Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Divalent siRNAs are bioavailable in the lung and efficiently block SARS-CoV-2 infection.

The continuous evolution of SARS-CoV-2 variants complicates efforts to combat the ongoing pandemic, underscoring the need for a dynamic platform for the rapid development of pan-viral variant therapeutics. Oligonucleotide therapeutics are enhancing the treatment of numerous diseases with unprecedented potency, duration of effect, and safety. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. Previous attempts to deliver therapeutic oligonucleotides to the lung have met with only modest success. Here, we report the development of a platform for identifying and generating potent, chemically modified multimeric siRNAs bioavailable in the lung after local intranasal and intratracheal delivery. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection and represent a new paradigm for antiviral therapeutic development for current and future pandemics.

SARS-CoV-2 Initiates Programmed Cell Death in Platelets.

[Figure: see text].

Pigment cell progenitor heterogeneity and reiteration of developmental signaling underlie melanocyte regeneration in zebrafish.

Tissue-resident stem and progenitor cells are present in many adult organs, where they are important for organ homeostasis and repair in response to injury. However, the signals that activate these cells and the mechanisms governing how these cells renew or differentiate are highly context-dependent and incompletely understood, particularly in non-hematopoietic tissues. In the skin, melanocyte stem and progenitor cells are responsible for replenishing mature pigmented melanocytes. In mammals, these cells reside in the hair follicle bulge and bulb niches where they are activated during homeostatic hair follicle turnover and following melanocyte destruction, as occurs in vitiligo and other skin hypopigmentation disorders. Recently, we identified melanocyte progenitors in adult zebrafish skin. To elucidate mechanisms governing melanocyte progenitor renewal and differentiation we analyzed individual transcriptomes from thousands of melanocyte lineage cells during the regeneration process. We identified transcriptional signatures for progenitors, deciphered transcriptional changes and intermediate cell states during regeneration, and analyzed cell-cell signaling changes to discover mechanisms governing melanocyte regeneration. We identified KIT signaling via the RAS/MAPK pathway as a regulator of melanocyte progenitor direct differentiation and asymmetric division. Our findings show how activation of different subpopulations of mitfa-positive cells underlies cellular transitions required to properly reconstitute the melanocyte pigmentary system following injury.

A Newly Engineered A549 Cell Line Expressing ACE2 and TMPRSS2 Is Highly Permissive to SARS-CoV-2, Including the Delta and Omicron Variants.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge, causing surges, breakthrough infections, and devastating losses-underscoring the importance of identifying SARS-CoV-2 antivirals. A simple, accessible human cell culture model permissive to SARS-CoV-2 variants is critical for identifying and assessing antivirals in a high-throughput manner. Although human alveolar A549 cells are a valuable model for studying respiratory virus infections, they lack two essential host factors for SARS-CoV-2 infection: angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). SARS-CoV-2 uses the ACE2 receptor for viral entry and TMPRSS2 to prime the SARS-CoV-2 spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a suitable human cell line for SARS-CoV-2 studies by transducing human ACE2 and TMPRSS2 into A549 cells. We show that subclones highly expressing ACE2 and TMPRSS2 ("ACE2plus" and the subclone "ACE2plusC3") are susceptible to infection with SARS-CoV-2, including the delta and omicron variants. These subclones express more ACE2 and TMPRSS2 transcripts than existing commercial A549 cells engineered to express ACE2 and TMPRSS2. Additionally, the antiviral drugs EIDD-1931, remdesivir, nirmatrelvir, and nelfinavir strongly inhibit SARS-CoV-2 variants in our infection model. Our data show that ACE2plusC3 cells are highly permissive to SARS-CoV-2 infection and can be used to identify anti-SARS-CoV-2 drugs.

Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION - Promising Strategies for Multiplex Identification of Viable Pathogens in Food.

Viable pathogenic bacteria are major biohazards that pose a significant threat to food safety. Despite the recent developments in detection platforms, multiplex identification of viable pathogens in food remains a major challenge. A novel strategy is developed through direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing on Nanopore MinION to achieve real-time multiplex identification of viable pathogens in food. Specifically, this study reports an optimized universal Nanopore sample extraction and library preparation protocol applicable to both Gram-positive and Gram-negative pathogenic bacteria, demonstrated using a cocktail culture of E. coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes, which were selected based on their impact on economic loss or prevalence in recent outbreaks. Further evaluation and validation confirmed the accuracy of direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing using Sanger sequencing and selective media. The study also included a comparison of different bioinformatic pipelines for metatranscriptomic and amplicon genomic analysis. MEGAN without rRNA mapping showed the highest accuracy of multiplex identification using the metatranscriptomic data. EPI2ME also demonstrated high accuracy using multiplex RT-PCR amplicon sequencing. In addition, a systemic comparison was drawn between Nanopore sequencing of the direct metatranscriptome RNA-seq and RT-PCR amplicons. Both methods are comparable in accuracy and time. Nanopore sequencing of RT-PCR amplicons has higher sensitivity, but Nanopore metatranscriptome sequencing excels in read length and dealing with complex microbiome and non-bacterial transcriptome backgrounds.