Mahyari, E. | bioRxiv Science

bioRxiv2023

The Human Infertility Single-cell Testis Atlas (HISTA): An interactive molecular scRNA-Seq reference of the human testis.

BackgroundThe Human Infertility Single-cell Testis Atlas (HISTA) is an interactive web tool and a reference for navigating the transcriptome of the human testis. It was developed using joint analyses of scRNA-Seq datasets derived from a dozen donors, including healthy adult controls, juveniles, and several infertility cases. HISTA is very different than other websites of testis scRNA-seq data, providing visualization and hypothesis testing tools on a batch-removed and integrated dataset of 23429 genes measured across 26093 cells using. ObjectiveThe main goal of this manuscript is to describe HISTA in detail and highlight its unique and novel features. MethodsTherefore, we used HISTA as a guide for its application and demonstrated HISTAs translational capacity to follow up on two observations of biological relevance. ResultsOur first analytical vignette identifies novel groupings of tightly regulated long non-coding RNA (lncRNA) molecules throughout spermatogenesis, suggesting specific functional genomics of these groupings. This analysis also found highly controlled expression of pairs of sense and antisense transcripts, suggesting conjoined regulatory mechanisms. In the next investigative vignette, we examined gene patterns in undifferentiated spermatogonia (USgs). We found the NANOS family of genes function as key drivers of transcriptomic signatures involved in human spermatogonial self-renewal programming; for the first time, demonstrating the relationship of NANOS1/2/3 transcripts in humans with scRNA-seq. Discussion and ConclusionsUsing HISTA, we found new observations that contribute to unraveling the mechanisms behind transcriptional regulation and maintenance germ cells across spermatogenesis. Furthermore, our findings provide guidance on future validation studies and experimental direction. Overall, HISTA continues to be utilized in testis-related research, and thus is updated regularly with new analytical methods, visualizations, and data. We aim to have it serve as a research environment for a broad range of investigators looking to explore the testis tissue and male infertility. Availability and ImplementationHISTA is available as an interactive web tool: https://conradlab.shinyapps.io/HISTA Source code and documentation for HISTA are provided on GitHub: https://github.com/eisascience/HISTA

bioinformatics↗

bioRxiv2020

In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the commonly used 68-1 strain of RhCMV has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptation that cause reduced viremia in RhCMV-naive animals and limited shedding compared to low passage isolates. Using sequence information from primary RhCMV isolates we constructed a full-length (FL) RhCMV by repairing all presumed mutations in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naive RM with the reconstituted virus resulted in significant replication in the blood similar to primary isolates of RhCMV and furthermore led to extensive viremia in many tissues at day 14 post infection. In contrast, viral dissemination and viremia was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques. Author SummaryHuman cytomegalovirus (HCMV) infections are generally asymptomatic in healthy immunocompetent individuals, but HCMV can cause serious disease after congenital infection and in individuals with immunocompromised immune systems. Since HCMV is highly species specific and cannot productively infect immunocompetent laboratory animals, experimental infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a closely related animal model for HCMV. By employing the unique ability of CMV to elicit robust and lasting cellular immunity, this model has also been instrumental in developing novel CMV-based vaccines against chronic and recurring infections with pathogens such as the human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (Mtb). However, most of this work was conducted with derivatives of the 68-1 strain of RhCMV which has acquired multiple genomic alterations in tissue culture. To model pathogenesis and immunology of clinical HCMV isolates we generated a full-length (FL) RhCMV clone representative of low passage isolates. Infection of RhCMV-naive RM with FL-RhCMV demonstrated viremia and tissue dissemination that was comparable to that of non-clonal low passage isolates. We further demonstrate that FL-RhCMV is strongly attenuated upon deletion of gene regions absent in 68-1 thus demonstrating the usefulness of FL-RhCMV to study RhCMV pathogenesis.

microbiology↗