Disorders of sex development (DSDs) are conditions affecting development of the gonads or genitalia. Variants in two key genes, SRY and its target SOX9, are an established cause of 46,XY DSD, but the genetic basis of many DSDs remains unknown. SRY-mediated SOX9 upregulation in the early gonad is crucial for testis development, yet the regulatory elements underlying this have not been identified in humans. Here, we identified four DSD patients with overlapping duplications or deletions upstream of SOX9. Bioinformatic analysis identified three putative enhancers for SOX9 that responded to different combinations of testis-specific regulators. All three enhancers showed synergistic activity and together drive SOX9 in the testis. This is the first study to identify SOX9 enhancers that, when duplicated or deleted, result in 46,XX or 46,XY sex reversal, respectively. These enhancers provide a hitherto missing link by which SRY activates SOX9 in humans, and establish SOX9 enhancer mutations as a significant cause of DSD.

The diversity of COVID-19 disease in otherwise healthy people, from seemingly asymptomatic infection to severe life-threatening disease, is not clearly understood. We passaged a naturally occurring near-ancestral SARS-CoV-2 variant, capable of infecting wild-type mice, and identified viral genomic mutations coinciding with the acquisition of severe disease in young adult mice and lethality in aged animals. Transcriptomic analysis of lung tissues from mice with severe disease elucidated a host antiviral response dominated mainly by interferon and IL-6 pathway activation in young mice, while in aged animals, a fatal outcome was dominated by TNF and TGF-β signaling. Congruent with our pathway analysis, we showed that young TNF-deficient mice had mild disease compared to controls and aged TNF-deficient animals were more likely to survive infection. Emerging clinical correlates of disease are consistent with our preclinical studies, and our model may provide value in defining aberrant host responses that are causative of severe COVID-19.

Polyadenylation is a dynamic process that is important in cellular physiology, which has implications in messenger RNA decay rates, translation efficiency, and isoform-specific regulation. Oxford Nanopore Technologies direct RNA sequencing provides a strategy for sequencing the full-length RNA molecule and analysis of the transcriptome. Several tools are currently available for poly(A) tail length estimation, including well-established methods like tailfindr and nanopolish, as well as more recent deep learning models like Dorado. However, there has been limited benchmarking of the accuracy of these tools against gold-standard datasets. In this article, we present our novel deep learning poly(A) estimation tool—BoostNano—and compare with 3 existing tools—tailfindr, nanopolish, and Dorado. We evaluate the 4 poly(A) estimation tools, using 2 sets of synthetic in vitro transcribed RNA standards with known poly(A) tail lengths—Sequin (30 or 60 nucleotides) and enhanced green fluorescent protein (10–150 nucleotides) RNA. Analyzing datasets with known ground-truth values is a valuable approach to measuring the accuracy of poly(A) length estimation. The tools demonstrated length- and sample-dependent performance, and accuracy was enhanced by averaging over multiple reads via estimation of the peak of the density distribution. Overall, Dorado is recommended as the preferred approach due to its relatively fast runtimes, low mean error, and ease of use with integration with base-calling. These results provide a reference for poly(A) tail length estimation analysis, aiding in improving our understanding of the transcriptome and the relationship between poly(A) tail length and other transcriptional mechanisms, including transcript stability or quantification.

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in feedlot cattle. Bovine herpesvirus-1 (BHV-1) is one of the main culprits of BRD; however, research on BHV-1 is hampered by the lack of suitable models for infection and drug testing. In this study, we established a novel bovine tracheal organoid culture grown in a basement membrane extract type 2 (BME2) matrix and compared it with the air–liquid interface (ALI) culture system. After differentiation, the matrix-embedded organoids developed beating cilia and demonstrated a transcriptomic profile similar to the ALI culture system. The matrix-embedded organoids were also highly susceptible to BHV-1 infection and immune stimulation by Pam₂Cys, an immunomodulator, which resulted in robust cytokine production and tracheal antimicrobial peptide mRNA upregulation. However, treatment of bovine tracheal organoid cultures with Pam₂Cys was not sufficient to inhibit viral infection or replication, suggesting a role of the non-epithelial cellular microenvironment in vivo.

Polyadenylation is a dynamic process which is important in cellular physiology. Oxford Nanopore Technologies direct RNA-sequencing provides a strategy for sequencing the full-length RNA molecule and analysis of the transcriptome and epi-transcriptome. There are currently several tools available for poly(A) tail-length estimation, including well-established tools such as tailfindr and nanopolish , as well as two more recent deep learning models: Dorado and BoostNano . However, there has been limited benchmarking of the accuracy of these tools against gold-standard datasets. In this paper we evaluate four poly(A) estimation tools using synthetic RNA standards (Sequins), which have known poly(A) tail-lengths and provide a valuable approach to measuring the accuracy of poly(A) tail-length estimation. All four tools generate mean tail-length estimates which lie within 12% of the correct value. Overall, Dorado is recommended as the preferred approach due to its relatively fast run times, low coefficient of variation and ease of use with integration with base-calling.