Effectiveness of Self-Collected, Ambient Temperature-Preserved Nasal Swabs Compared to Samples Collected by Trained Staff for Genotyping of Respiratory Viruses by Shotgun RNA Sequencing: Comparative Study.

BACKGROUND: The SARS-CoV-2 pandemic has underscored the need for field specimen collection and transport to diagnostic and public health laboratories. Self-collected nasal swabs transported without dependency on a cold chain have the potential to remove critical barriers to testing, expand testing capacity, and reduce opportunities for exposure of health professionals in the context of a pandemic. OBJECTIVE: We compared nasal swab collection by study participants from themselves and their children at home to collection by trained research staff. METHODS: Each adult participant collected 1 nasal swab, sampling both nares with the single swab, after which they collected 1 nasal swab from 1 child. After all the participant samples were collected for the household, the research staff member collected a separate single duplicate sample from each individual. Immediately after the sample collection, the adult participants completed a questionnaire about the acceptability of the sampling procedures. Swabs were placed in temperature-stable preservative and respiratory viruses were detected by shotgun RNA sequencing, enabling viral genome analysis. RESULTS: In total, 21 households participated in the study, each with 1 adult and 1 child, yielding 42 individuals with paired samples. Study participants reported that self-collection was acceptable. Agreement between identified respiratory viruses in both swabs by RNA sequencing demonstrated that adequate collection technique was achieved by brief instructions. CONCLUSIONS: Our results support the feasibility of a scalable and convenient means for the identification of respiratory viruses and implementation in pandemic preparedness for novel respiratory pathogens.

Fecal Host Transcriptomics for Non-Invasive Human Mucosal Immune Profiling: Proof of Concept in Clostridium Difficile Infection.

Background: Host factors play an important role in pathogenesis and disease outcome in Clostridium difficile infection (CDI), and characterization of these responses could uncover potential host biomarkers to complement existing microbe-based diagnostics. Methods: We extracted RNA from fecal samples of patients with CDI and profiled human mRNA using amplicon-based next-generation sequencing (NGS). We compared the fecal host mRNA transcript expression profiles of patients with CDI to controls with non-CDI diarrhea. Results: We found that the ratio of human actin gamma 1 (ACTG1) to 16S ribosomal RNA (rRNA) was highly correlated with NGS quality as measured by percentage of reads on target. Patients with CDI could be differentiated from those with non-CDI diarrhea based on their fecal mRNA expression profiles using principal component analysis. Among the most differentially expressed genes were ones related to immune response (IL23A, IL34) and actin-cytoskeleton function (TNNT1, MYL4, SMTN, MYBPC3, all adjusted P-values < 1 × 10-3). Conclusions: In this proof-of-concept study, we used host fecal transcriptomics for non-invasive profiling of the mucosal immune response in CDI. We identified differentially expressed genes with biological plausibility based on animal and cell culture models. This demonstrates the potential of fecal transcriptomics to uncover host-based biomarkers for enteric infections.

Gestational Psittacosis Resulting in Neonatal Death Identified by Next-Generation RNA Sequencing of Postmortem, Formalin-Fixed Lung Tissue.

Psittacosis is a rare zoonosis that can cause severe disease and adverse outcomes during pregnancy. We identified a previously elusive case of psittacosis causing premature delivery and infant death by next-generation RNA sequencing of postmortem tissues. Hypothesis-free pathogen detection in postmortem specimens can increase the yield of epidemiologic and cause-of-death studies.

Pask integrates hormonal signaling with histone modification via Wdr5 phosphorylation to drive myogenesis.

PAS domain containing protein kinase (Pask) is an evolutionarily conserved protein kinase implicated in energy homeostasis and metabolic regulation across eukaryotic species. We now describe an unexpected role of Pask in promoting the differentiation of myogenic progenitor cells, embryonic stem cells and adipogenic progenitor cells. This function of Pask is dependent upon its ability to phosphorylate Wdr5, a member of several protein complexes including those that catalyze histone H3 Lysine 4 trimethylation (H3K4me3) during transcriptional activation. Our findings suggest that, during myoblast differentiation, Pask stimulates the conversion of repressive H3K4me1 to activating H3K4me3 marks on the promoter of the differentiation gene myogenin (Myog) via Wdr5 phosphorylation. This enhances accessibility of the MyoD transcription factor and enables transcriptional activation of the Myog promoter to initiate muscle differentiation. Thus, as an upstream kinase of Wdr5, Pask integrates signaling cues with the transcriptional network to regulate the differentiation of progenitor cells.

Dpp-induced Egfr signaling triggers postembryonic wing development in Drosophila.

The acquisition of flight contributed to the success of insects and winged forms are present in most orders. Key to understanding the origin of wings will be knowledge of the earliest postembryonic events promoting wing outgrowth. The Drosophila melanogaster wing is intensely studied as a model appendage, and yet little is known about the beginning of wing outgrowth. Vein (Vn) is a neuregulin-like ligand for the EGF receptor (Egfr), which is necessary for global development of the early Drosophila wing disc. vn is not expressed in the embryonic wing primordium and thus has to be induced de novo in the nascent larval wing disc. We find that Decapentaplegic (Dpp), a Bone Morphogenetic Protein (BMP) family member, provides the instructive signal for initiating vn expression. The signaling involves paracrine communication between two epithelia in the early disc. Once initiated, vn expression is amplified and maintained by autocrine signaling mediated by the E-twenty six (ETS)-factor PointedP2 (PntP2). This interplay of paracrine and autocrine signaling underlies the spatial and temporal pattern of induction of Vn/Egfr target genes and explains both body wall development and wing outgrowth. It is possible this gene regulatory network governing expression of an EGF ligand is conserved and reflects a common origin of insect wings.

The ETS gene ETV4 is required for anchorage-independent growth and a cell proliferation gene expression program in PC3 prostate cells.

Chromosomal abnormalities that give rise to elevated expression levels of the ETS genes ETV1, ETV4, ETV5, or ERG are prevalent in prostate cancer, but the function of these transcription factors in carcinogenesis is not clear. Previous work in cell lines implicates ERG, ETV1, and ETV5 as regulators of invasive growth but not transformation. Here we show that the PC3 prostate cancer cell line provides a model system to study the over-expression of ETV4. Migration assays, anchorage independent growth assays, and microarray analysis indicate that high ETV4 expression contributes to both transformation and cellular motility in PC3 cells. ETV4 directly bound the 5' and 3' MYC enhancers and modulated expression of both MYC and other cell proliferation genes, demonstrating a potential role in cell growth control. Despite this novel role for ETV4 in anchorage independent growth, ETV4 over-expression in normal prostate-derived RWPE-1 cells showed effects similar to ETV1 over-expression - increased cellular motility, and an up-regulation of genes encoding extracellular proteins as well as ones important for development, inflammation, and wound healing. Because ETV1 and ETV4 have similar roles when introduced to the same cellular background, we suggest that the requirement of high ETV4 expression for maintenance of the anchorage-independent growth in PC3 cells is due to a specific characteristic of this cell line rather than a function of ETV4 that is distinct from the other oncogenic ETS genes. Thus, the function of ETS genes in prostate cancer may differ based on other genetic alterations in a tumor.

Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes.

Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene Ras(V12) (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of Ras(V12) is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.

Functional analysis of genes differentially expressed in the Drosophila wing disc: role of transcripts enriched in the wing region.

Differential gene expression is the major mechanism underlying the development of specific body regions. Here we assessed the role of genes differentially expressed in the Drosophila wing imaginal disc, which gives rise to two distinct adult structures: the body wall and the wing. Reverse genetics was used to test the function of uncharacterized genes first identified in a microarray screen as having high levels of expression in the presumptive wing. Such genes could participate in elaborating the specific morphological characteristics of the wing. The activity of the genes was modulated using misexpression and RNAi-mediated silencing. Misexpression of eight of nine genes tested caused phenotypes. Of 12 genes tested, 10 showed effective silencing with RNAi transgenes, but only 3 of these had resulting phenotypes. The wing phenotypes resulting from RNAi suggest that CG8780 is involved in patterning the veins in the proximal region of the wing blade and that CG17278 and CG30069 are required for adhesion of wing surfaces. Venation and apposition of the wing surfaces are processes specific to wing development providing a correlation between the expression and function of these genes. The results show that a combination of expression profiling and tissue-specific gene silencing has the potential to identify new genes involved in wing development and hence to contribute to our understanding of this process. However, there are both technical and biological limitations to this approach, including the efficacy of RNAi and the role that gene redundancy may play in masking phenotypes.

Prepattern genes and signaling molecules regulate stripe expression to specify Drosophila flight muscle attachment sites.

In Drosophila, muscles attach to epidermal tendon cells specified by the gene stripe (sr). Flight muscle attachment sites are prefigured on the wing imaginal disc by sr expression in discrete domains. We describe the mechanisms underlying the specification of these domains of sr expression. We show that the concerted activities of the wingless (wg), decapentaplegic (dpp) and Notch (N) signaling pathways, and the prepattern genes pannier (pnr) and u-shaped (ush) establish domains of sr expression. N is required for initiation of sr expression. pnr is a positive regulator of sr, and is inhibited by ush in this function. The Wg signal differentially influences the formation of different sr domains. These results identify the multiple regulatory elements involved in the positioning of Drosophila flight muscle attachment sites.