Next Generation Sequencing of the Pig αß TCR Repertoire Identifies the Porcine Invariant NKT Cell Receptor.

Swine represent the only livestock with an established invariant NKT (iNKT) cell-CD1d system. In this study, we exploited the fact that pig iNKT cells can be purified using a mouse CD1d tetramer reagent to establish their TCR repertoire by next generation sequencing. CD1d tetramer-positive pig cells predominantly expressed an invariant Vα-Jα rearrangement, without nontemplate nucleotide diversity, homologous to the Vα24-Jα18 and Vα14-Jα18 rearrangements of human and murine iNKT cells. The coexpressed ß-chain used a Vß segment homologous to the semivariant Vß11 and Vß8.2 segments of human and murine iNKT cell receptors. Molecular modeling found that contacts within CD1d and CDR1α that underlie fine specificity differences between mouse and human iNKT cells are conserved between pigs and humans, indicating that the response of porcine and human iNKT cells to CD1d-restricted Ags may be similar. Accordingly, pigs, which are an important species for diverse fields of biomedical research, may be useful for developing human-based iNKT cell therapies for cancer, infectious diseases, and other disorders. Our study also sequenced the expressed TCR repertoire of conventional porcine αß T cells, which identified 48 Vα, 50 Jα, 18 Vß, and 18 Jß sequences, most of which correspond to human gene segments. These findings provide information on the αß TCR usage of pigs, which is understudied and deserves further attention.

YES1 amplification is a mechanism of acquired resistance to EGFR inhibitors identified by transposon mutagenesis and clinical genomics.

In ∼30% of patients with EGFR-mutant lung adenocarcinomas whose disease progresses on EGFR inhibitors, the basis for acquired resistance remains unclear. We have integrated transposon mutagenesis screening in an EGFR-mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify mechanisms of resistance to EGFR inhibitors. The most prominent candidate genes identified by insertions in or near the genes during the screen were MET, a gene whose amplification is known to mediate resistance to EGFR inhibitors, and the gene encoding the Src family kinase YES1. Cell clones with transposon insertions that activated expression of YES1 exhibited resistance to all three generations of EGFR inhibitors and sensitivity to pharmacologic and siRNA-mediated inhibition of YES1 Analysis of clinical genomic sequencing data from cases of acquired resistance to EGFR inhibitors revealed amplification of YES1 in five cases, four of which lacked any other known mechanisms of resistance. Preinhibitor samples, available for two of the five patients, lacked YES1 amplification. None of 136 postinhibitor samples had detectable amplification of other Src family kinases (SRC and FYN). YES1 amplification was also found in 2 of 17 samples from ALK fusion-positive lung cancer patients who had progressed on ALK TKIs. Taken together, our findings identify acquired amplification of YES1 as a recurrent and targetable mechanism of resistance to EGFR inhibition in EGFR-mutant lung cancers and demonstrate the utility of transposon mutagenesis in discovering clinically relevant mechanisms of drug resistance.

Analysis of Mitochondrial DNA Polymorphisms in the Human Cell Lines HepaRG and SJCRH30.

The mitochondrial DNA (mtDNA) sequences of two commonly used human cell lines, HepaRG and SJCRH30, were determined. HepaRG originates from a liver tumor obtained from a patient with hepatocarcinoma and hepatitis C while SJCRH30 originates from a rhabdomyosarcoma patient tumor. In comparison to the revised Cambridge Reference Sequence, HepaRG and SJCRH30 mtDNA each contain 14 nucleotide variations. In addition to an insertion of a cytosine at position 315 (315insC), the mtDNA sequences from both cell types share six common polymorphisms. Heteroplasmic variants were identified in both cell types and included the identification of the 315insC mtDNA variant at 42 and 75% heteroplasmy in HepaRG and SJCRH30, respectively. Additionally, a novel heteroplasmic G13633A substitution in the HepaRG ND5 gene was detected at 33%. Previously reported cancer-associated mtDNA variants T195C and T16519C were identified in SJCRH30, both at homoplasmy (100%), while HepaRG mtDNA harbors a known prostate cancer-associated T6253C substitution at near homoplasmy, 95%. Based on our sequencing analysis, HepaRG mtDNA is predicted to lie within haplogroup branch H15a1 while SJCRH30 mtDNA is predicted to localize to H27c. The catalog of polymorphisms and heteroplasmy reported here should prove useful for future investigations of mtDNA maintenance in HepaRG and SJCRH30 cell lines.

Stable heteroplasmy at the single-cell level is facilitated by intercellular exchange of mtDNA.

Eukaryotic cells carry two genomes, nuclear (nDNA) and mitochondrial (mtDNA), which are ostensibly decoupled in their replication, segregation and inheritance. It is increasingly appreciated that heteroplasmy, the occurrence of multiple mtDNA haplotypes in a cell, plays an important biological role, but its features are not well understood. Accurately determining the diversity of mtDNA has been difficult, due to the relatively small amount of mtDNA in each cell (<1% of the total DNA), the intercellular variability of mtDNA content and mtDNA pseudogenes (Numts) in nDNA. To understand the nature of heteroplasmy, we developed Mseek, a novel technique to purify and sequence mtDNA. Mseek yields high purity (>90%) mtDNA and its ability to detect rare variants is limited only by sequencing depth, providing unprecedented sensitivity and specificity. Using Mseek, we confirmed the ubiquity of heteroplasmy by analyzing mtDNA from a diverse set of cell lines and human samples. Applying Mseek to colonies derived from single cells, we find heteroplasmy is stably maintained in individual daughter cells over multiple cell divisions. We hypothesized that the stability of heteroplasmy could be facilitated by intercellular exchange of mtDNA. We explicitly demonstrate this exchange by co-culturing cell lines with distinct mtDNA haplotypes. Our results shed new light on the maintenance of heteroplasmy and provide a novel platform to investigate features of heteroplasmy in normal and diseased states.

High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries.

We introduce two large-scale resources for functional analysis of microRNA (miRNA): a decoy library for inhibiting miRNA function and a sensor library for monitoring microRNA activity. To take advantage of the sensor library, we developed a high-throughput assay called Sensor-seq to simultaneously quantify the activity of hundreds of miRNAs. Using this approach, we show that only the most abundant miRNAs in a cell mediate target suppression. Over 60% of detected miRNAs had no discernible activity, which indicated that the functional 'miRNome' of a cell is considerably smaller than currently inferred from profiling studies. Moreover, some highly expressed miRNAs exhibited relatively weak activity, which in some cases correlated with a high target-to-miRNA ratio or increased nuclear localization of the miRNA. Finally, we show that the miRNA decoy library can be used for pooled loss-of-function studies. These tools are valuable resources for studying miRNA biology and for miRNA-based therapeutics.

MiST: a new approach to variant detection in deep sequencing datasets.

MiST is a novel approach to variant calling from deep sequencing data, using the inverted mapping approach developed for Geoseq. Reads that can map to a targeted exonic region are identified using exact matches to tiles from the region. The reads are then aligned to the targets to discover variants. MiST carefully handles paralogous reads that map ambiguously to the genome and clonal reads arising from PCR bias, which are the two major sources of errors in variant calling. The reduced computational complexity of mapping selected reads to targeted regions of the genome improves speed, specificity and sensitivity of variant detection. Compared with variant calls from the GATK platform, MiST showed better concordance with SNPs from dbSNP and genotypes determined by an exonic-SNP array. Variant calls made only by MiST confirm at a high rate (>90%) by Sanger sequencing. Thus, MiST is a valuable alternative tool to analyse variants in deep sequencing data.

Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing.

Deep sequencing of small RNAs (sRNA-seq) is now the gold standard for small RNA profiling and discovery. Biases in sRNA-seq have been reported, but their etiology remains unidentified. Through a comprehensive series of sRNA-seq experiments, we establish that the predominant cause of the bias is the RNA ligases. We further demonstrate that RNA ligases have strong sequence-specific biases which distort the small RNA profiles considerably. We have devised a pooled adapter strategy to overcome this bias, and validated the method through data derived from microarray and qPCR. In light of our findings, published small RNA profiles, as well as barcoding strategies using adapter-end modifications, may need to be revisited. Importantly, by providing a wide spectrum of substrate for the ligase, the pooled-adapter strategy developed here provides a means to overcome issues of bias, and generate more accurate small RNA profiles.

Marburg and Ebola Virus Infections Elicit a Complex, Muted Inflammatory State in Bats.

The Marburg and Ebola filoviruses cause a severe, often fatal, disease in humans and nonhuman primates but have only subclinical effects in bats, including Egyptian rousettes, which are a natural reservoir of Marburg virus. A fundamental question is why these viruses are highly pathogenic in humans but fail to cause disease in bats. To address this question, we infected one cohort of Egyptian rousette bats with Marburg virus and another cohort with Ebola virus and harvested multiple tissues for mRNA expression analysis. While virus transcripts were found primarily in the liver, principal component analysis (PCA) revealed coordinated changes across multiple tissues. Gene signatures in kidney and liver pointed at induction of vasodilation, reduction in coagulation, and changes in the regulation of iron metabolism. Signatures of immune response detected in spleen and liver indicated a robust anti-inflammatory state signified by macrophages in the M2 state and an active T cell response. The evolutionary divergence between bats and humans of many responsive genes might provide a framework for understanding the differing outcomes upon infection by filoviruses. In this study, we outline multiple interconnected pathways that respond to infection by MARV and EBOV, providing insights into the complexity of the mechanisms that enable bats to resist the disease caused by filoviral infections. The results have the potential to aid in the development of new strategies to effectively mitigate and treat the disease caused by these viruses in humans.

Accessible LAMP-Enabled Rapid Test (ALERT) for Detecting SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. Polymerase chain reaction (PCR)-based diagnostic tests are expensive, reliant on centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests that bind and detect the surface proteins of a virus are rapid and scalable but suffer from high false negative rates. To address this problem, an inexpensive, simple, and robust 60-minute do-it-yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/µL) and specificity (>97% true negative rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed. ALERT (Accessible LAMP-Enabled Rapid Test) incorporates the following features: (1) increased shelf-life and ambient temperature storage, compared to liquid reaction mixes, by using wax layers to isolate enzymes from other reagents; (2) improved specificity compared to other LAMP end-point reporting methods, by using sequence-specific QUASR (quenching of unincorporated amplification signal reporters); (3) increased sensitivity, compared to methods without purification through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal; (4) quality control with a nasopharyngeal-specific mRNA target; and (5) co-detection of other respiratory viruses, such as influenza B, by multiplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for labs and hospitals. With minimal effort, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms, or nucleic acid-based markers.

Transposon mutagenesis in Mycobacterium kansasii links a small RNA gene to colony morphology and biofilm formation and identifies 9,885 intragenic insertions that do not compromise colony outgrowth.

Mycobacterium kansasii (Mk) is a resilient opportunistic human pathogen that causes tuberculosis-like chronic pulmonary disease and mortality stemming from comorbidities and treatment failure. The standard treatment of Mk infections requires costly, long-term, multidrug courses with adverse side effects. The emergence of drug-resistant isolates further complicates the already challenging drug therapy regimens and threatens to compromise the future control of Mk infections. Despite the increasingly recognized global burden of Mk infections, the biology of this opportunistic pathogen remains essentially unexplored. In particular, studies reporting gene function or generation of defined mutants are scarce. Moreover, no transposon (Tn) mutagenesis tool has been validated for use in Mk, a situation limiting the repertoire of genetic approaches available to accelerate the dissection of gene function and the generation of gene knockout mutants in this poorly characterized pathogen. In this study, we validated the functionality of a powerful Tn mutagenesis tool in Mk and used this tool in conjunction with a forward genetic screen to establish a previously unrecognized role of a conserved mycobacterial small RNA gene of unknown function in colony morphology features and biofilm formation. We also combined Tn mutagenesis with next-generation sequencing to identify 12,071 Tn insertions that do not compromise viability in vitro. Finally, we demonstrated the susceptibility of the Galleria mellonella larva to Mk, setting the stage for further exploration of this simple and economical infection model system to the study of this pathogen.

Deep sequencing of mRNA in CD24(-) and CD24(+) mammary carcinoma Mvt1 cell line.

CD24 is an anchored cell surface marker that is highly expressed in cancer cells (Lee et al., 2009) and its expression is associated with poorer outcome of cancer patients (Kristiansen et al., 2003). Phenotype comparison between two subpopulations derived from the Mvt1 cell line, CD24(-) cells (with no CD24 cell surface expression) and the CD24(+) cells, identified high tumorigenic capacity for the CD24(+) cells. In order to reveal the transcripts that support the CD24(+) aggressive and invasive phenotype we compared the gene profiles of these two subpopulations. mRNA profiles of CD24(-) and CD24(+) cells were generated by deep sequencing, in triplicate, using an Illumina HiSeq 2500. Here we provide a detailed description of the mRNA-seq analysis from our recent study (Rostoker et al., 2015). The mRNA-seq data have been deposited in the NCBI GEO database (accession number GSE68746).

Tbx3 Controls Dppa3 Levels and Exit from Pluripotency toward Mesoderm.

Tbx3, a member of the T-box family, plays important roles in development, stem cells, nuclear reprogramming, and cancer. Loss of Tbx3 induces differentiation in mouse embryonic stem cells (mESCs). However, we show that mESCs exist in an alternate stable pluripotent state in the absence of Tbx3. In-depth transcriptome analysis of this mESC state reveals Dppa3 as a direct downstream target of Tbx3. Also, Tbx3 facilitates the cell fate transition from pluripotent cells to mesoderm progenitors by directly repressing Wnt pathway members required for differentiation. Wnt signaling regulates differentiation of mESCs into mesoderm progenitors and helps to maintain a naive pluripotent state. We show that Tbx3, a downstream target of Wnt signaling, fine tunes these divergent roles of Wnt signaling in mESCs. In conclusion, we identify a signaling-TF axis that controls the exit of mESCs from a self-renewing pluripotent state toward mesoderm differentiation.

Kinetic analysis reveals the fate of a microRNA following target regulation in mammalian cells.

Considerable details about microRNA (miRNA) biogenesis and regulation have been uncovered, but little is known about the fate of the miRNA subsequent to target regulation. To gain insight into this process, we carried out kinetic analysis of a miRNA's turnover following termination of its biogenesis and during regulation of a target that is not subject to Ago2-mediated catalytic cleavage. By quantitating the number of molecules of the miRNA and its target in steady state and in the course of its decay, we found that each miRNA molecule was able to regulate at least two target transcripts, providing in vivo evidence that the miRNA is not irreversibly sequestered with its target and that the nonslicing pathway of miRNA regulation is multiple-turnover. Using deep sequencing, we further show that miRNA recycling is limited by target regulation, which promotes posttranscriptional modifications to the 3' end of the miRNA and accelerates the miRNA's rate of decay. These studies provide new insight into the efficiency of miRNA regulation that help to explain how a miRNA can regulate a vast number of transcripts and that identify one of the mechanisms that impart specificity to miRNA decay in mammalian cells.