Circulating mitochondrial DNA is a proinflammatory DAMP in sickle cell disease.

The pathophysiology of sickle cell disease (SCD) is driven by chronic inflammation fueled by damage associated molecular patterns (DAMPs). We show that elevated cell-free DNA (cfDNA) in patients with SCD is not just a prognostic biomarker, it also contributes to the pathological inflammation. Within the elevated cfDNA, patients with SCD had a significantly higher ratio of cell-free mitochondrial DNA (cf-mtDNA)/cell-free nuclear DNA compared with healthy controls. Additionally, mitochondrial DNA in patient samples showed significantly disproportionately increased hypomethylation compared with healthy controls, and it was increased further in crises compared with steady-state. Using flow cytometry, structured illumination microscopy, and electron microscopy, we showed that circulating SCD red blood cells abnormally retained their mitochondria and, thus, are likely to be the source of the elevated cf-mtDNA in patients with SCD. Patient plasma containing high levels of cf-mtDNA triggered the formation of neutrophil extracellular traps (NETs) that was substantially reduced by inhibition of TANK-binding kinase 1, implicating activation of the cGAS-STING pathway. cf-mtDNA is an erythrocytic DAMP, highlighting an underappreciated role for mitochondria in sickle pathology. These trials were registered at www.clinicaltrials.gov as #NCT00081523, #NCT03049475, and #NCT00047996.

Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques.

The programmable nuclease technology CRISPR-Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR-Cas9 therapeutically. Here, we utilized a rhesus macaque model to compare the predictive values of CIRCLE-seq, an in vitro off-target prediction method, with in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-seq and ISP for a CD33 guide RNA (gRNA) with thousands of off-target sites predicted by ISP and CIRCLE-seq. We found poor correlation between the sites predicted by the two methods. When almost 500 sites predicted by each method were analyzed by error-corrected sequencing of hematopoietic cells following transplantation, 19 off-target sites revealed insertion or deletion mutations. Of these sites, 8 were predicted by both methods, 8 by CIRCLE-seq only, and 3 by ISP only. The levels of cells with these off-target edits exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In addition, we utilized an unbiased method termed CAST-seq to search for translocations between the on-target site and off-target sites present in animals following transplantation, detecting one specific translocation that persisted in blood cells for at least 1 year following transplantation. In conclusion, neither CIRCLE-seq or ISP predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods, may be required for optimizing safety of clinical development.

Cell-Free DNA to Detect Heart Allograft Acute Rejection.

BACKGROUND: After heart transplantation, endomyocardial biopsy (EMBx) is used to monitor for acute rejection (AR). Unfortunately, EMBx is invasive, and its conventional histological interpretation has limitations. This is a validation study to assess the performance of a sensitive blood biomarker-percent donor-derived cell-free DNA (%ddcfDNA)-for detection of AR in cardiac transplant recipients. METHODS: This multicenter, prospective cohort study recruited heart transplant subjects and collected plasma samples contemporaneously with EMBx for %ddcfDNA measurement by shotgun sequencing. Histopathology data were collected to define AR, its 2 phenotypes (acute cellular rejection [ACR] and antibody-mediated rejection [AMR]), and controls without rejection. The primary analysis was to compare %ddcfDNA levels (median and interquartile range [IQR]) for AR, AMR, and ACR with controls and to determine %ddcfDNA test characteristics using receiver-operator characteristics analysis. RESULTS: The study included 171 subjects with median posttransplant follow-up of 17.7 months (IQR, 12.1-23.6), with 1392 EMBx, and 1834 %ddcfDNA measures available for analysis. Median %ddcfDNA levels decayed after surgery to 0.13% (IQR, 0.03%-0.21%) by 28 days. Also, %ddcfDNA increased again with AR compared with control values (0.38% [IQR, 0.31-0.83%], versus 0.03% [IQR, 0.01-0.14%]; P<0.001). The rise was detected 0.5 and 3.2 months before histopathologic diagnosis of ACR and AMR. The area under the receiver operator characteristic curve for AR was 0.92. A 0.25%ddcfDNA threshold had a negative predictive value for AR of 99% and would have safely eliminated 81% of EMBx. In addition, %ddcfDNA showed distinctive characteristics comparing AMR with ACR, including 5-fold higher levels (AMR ≥2, 1.68% [IQR, 0.49-2.79%] versus ACR grade ≥2R, 0.34% [IQR, 0.28-0.72%]), higher area under the receiver operator characteristic curve (0.95 versus 0.85), higher guanosine-cytosine content, and higher percentage of short ddcfDNA fragments. CONCLUSIONS: We found that %ddcfDNA detected AR with a high area under the receiver operator characteristic curve and negative predictive value. Monitoring with ddcfDNA demonstrated excellent performance characteristics for both ACR and AMR and led to earlier detection than the EMBx-based monitoring. This study supports the use of %ddcfDNA to monitor for AR in patients with heart transplant and paves the way for a clinical utility study. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02423070.

Activation and inhibition of nonsense-mediated mRNA decay control the abundance of alternative polyadenylation products.

Alternative polyadenylation (APA) produces transcript 3' untranslated regions (3'UTRs) with distinct sequences, lengths, stabilities and functions. We show here that APA products include a class of cryptic nonsense-mediated mRNA decay (NMD) substrates with extended 3'UTRs that gene- or transcript-level analyses of NMD often fail to detect. Transcriptome-wide, the core NMD factor UPF1 preferentially recognizes long 3'UTR products of APA, leading to their systematic downregulation. Counteracting this mechanism, the multifunctional RNA-binding protein PTBP1 regulates the balance of short and long 3'UTR isoforms by inhibiting NMD, in addition to its previously described modulation of co-transcriptional polyadenylation (polyA) site choice. Further, we find that many transcripts with altered APA isoform abundance across multiple tumor types are controlled by NMD. Together, our findings reveal a widespread role for NMD in shaping the outcomes of APA.

Properties of global- and local-ancestry adjustments in genetic association tests in admixed populations.

Population substructure can lead to confounding in tests for genetic association, and failure to adjust properly can result in spurious findings. Here we address this issue of confounding by considering the impact of global ancestry (average ancestry across the genome) and local ancestry (ancestry at a specific chromosomal location) on regression parameters and relative power in ancestry-adjusted and -unadjusted models. We examine theoretical expectations under different scenarios for population substructure; applying different regression models, verifying and generalizing using simulations, and exploring the findings in real-world admixed populations. We show that admixture does not lead to confounding when the trait locus is tested directly in a single admixed population. However, if there is more complex population structure or a marker locus in linkage disequilibrium (LD) with the trait locus is tested, both global and local ancestry can be confounders. Additionally, we show the genotype parameters of adjusted and unadjusted models all provide tests for LD between the marker and trait locus, but in different contexts. The local ancestry adjusted model tests for LD in the ancestral populations, while tests using the unadjusted and the global ancestry adjusted models depend on LD in the admixed population(s), which may be enriched due to different ancestral allele frequencies. Practically, this implies that global-ancestry adjustment should be used for screening, but local-ancestry adjustment may better inform fine mapping and provide better effect estimates at trait loci.

Targeted RNA-sequencing for the quantification of measurable residual disease in acute myeloid leukemia.

Great effort is spent on developing therapies to improve the dire outcomes of those diagnosed with acute myeloid leukemia. The methods for quantifying response to therapeutic intervention have however lacked sensitivity. Patients achieving a complete remission as defined by conventional cytomorphological methods therefore remain at risk of subsequent relapse due to disease persistence. Improved risk stratification is possible based on tests designed to detect this residual leukemic burden (measurable residual disease). However, acute myeloid leukemia is a genetically diverse set of diseases, which has made it difficult to develop a single, highly reproducible, and sensitive assay for measurable residual disease. Here we present the development of a digital targeted RNA-sequencing-based approach designed to overcome these limitations by detecting all newly approved European LeukemiaNet molecular targets for measurable residual disease in acute myeloid leukemia in a single standardized assay. Iterative modifications and novel bioinformatics approaches resulted in a greater than 100-fold increase in performance compared with commercially available targeted RNA-sequencing approaches and a limit of detection as low as one leukemic cell in 100,000 cells measured, which is comparable to quantitative polymerase chain reaction analysis, the current gold standard for the detection of measurable residual disease. This assay, which can be customized and expanded, is the first demonstrated use of high-sensitivity RNA-sequencing for measurable residual disease detection in acute myeloid leukemia and could serve as a broadly applicable standardized tool.

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers.

Nonhuman primate (NHP) induced pluripotent stem cells (iPSCs) offer the opportunity to investigate the safety, feasibility, and efficacy of proposed iPSC-derived cellular delivery in clinically relevant in vivo models. However, there is need for stable, robust, and safe labeling methods for NHP iPSCs and their differentiated lineages to study survival, proliferation, tissue integration, and biodistribution following transplantation. Here we investigate the utility of the adeno-associated virus integration site 1 (AAVS1) as a safe harbor for the addition of transgenes in our rhesus macaque iPSC (RhiPSC) model. A clinically relevant marker gene, human truncated CD19 (hΔCD19), or GFP was inserted into the AAVS1 site in RhiPSCs using the CRISPR/Cas9 system. Genetically modified RhiPSCs maintained normal karyotype and pluripotency, and these clones were able to further differentiate into all three germ layers in vitro and in vivo. In contrast to transgene delivery using randomly integrating viral vectors, AAVS1 targeting allowed stable transgene expression following differentiation. Off-target mutations were observed in some edited clones, highlighting the importance of careful characterization of these cells prior to downstream applications. Genetically marked RhiPSCs will be useful to further advance clinically relevant models for iPSC-based cell therapies.

Kinetics of phytosterol metabolism in neonates receiving parenteral nutrition.

BACKGROUND: Phytosterols in soybean oil (SO) lipids likely contribute to parenteral nutrition-associated liver disease (PNALD) in infants. No characterization of phytosterol metabolism has been done in infants receiving SO lipids. METHODS: In a prospective cohort study, 45 neonates (36 SO lipid vs. 9 control) underwent serial blood sample measurements of sitosterol, campesterol, and stigmasterol. Mathematical modeling was used to determine pharmacokinetic parameters of phytosterol metabolism and phytosterol exposure. RESULTS: Compared to controls, SO lipid-exposed infants had significantly higher levels of sitosterol and campesterol (P < 0.01). During SO lipid infusion, sitosterol and campesterol reached half of steady-state plasma levels within 1.5 and 0.8 d, respectively. Steady-state level was highest for sitosterol (1.68 mg/dl), followed by campesterol (0.98 mg/dl), and lowest for stigmasterol (0.01 mg/dl). Infants born < 28 wk gestational age had higher sitosterol steady-state levels (P = 0.03) and higher area under the curve for sitosterol (P = 0.03) during the first 5 d of SO lipid (AUC5) than infants born ≥ 28 wk gestational age. CONCLUSION: Phytosterols in SO lipid accumulate rapidly in neonates. Very preterm infants receiving SO lipid have higher sitosterol exposure, and may have poorly developed mechanisms of eliminating phytosterols that may contribute to their vulnerability to PNALD.

Genetically defined individual reference ranges for tryptase limit unnecessary procedures and unmask myeloid neoplasms.

Serum tryptase is a biomarker used to aid in the identification of certain myeloid neoplasms, most notably systemic mastocytosis, where basal serum tryptase (BST) levels >20 ng/mL are a minor criterion for diagnosis. Although clonal myeloid neoplasms are rare, the common cause for elevated BST levels is the genetic trait hereditary α-tryptasemia (HαT) caused by increased germline TPSAB1 copy number. To date, the precise structural variation and mechanism(s) underlying elevated BST in HαT and the general clinical utility of tryptase genotyping, remain undefined. Through cloning, long-read sequencing, and assembling of the human tryptase locus from an individual with HαT, and validating our findings in vitro and in silico, we demonstrate that BST elevations arise from overexpression of replicated TPSAB1 loci encoding canonical α-tryptase protein owing to coinheritance of a linked overactive promoter element. Modeling BST levels based on TPSAB1 replication number, we generate new individualized clinical reference values for the upper limit of normal. Using this personalized laboratory medicine approach, we demonstrate the clinical utility of tryptase genotyping, finding that in the absence of HαT, BST levels >11.4 ng/mL frequently identify indolent clonal mast cell disease. Moreover, substantial BST elevations (eg, >100 ng/mL), which would ordinarily prompt bone marrow biopsy, can result from TPSAB1 replications alone and thus be within normal limits for certain individuals with HαT.

The PPR domain of mitochondrial RNA polymerase is an exoribonuclease required for mtDNA replication in Drosophila melanogaster.

Mitochondrial DNA (mtDNA) replication and transcription are of paramount importance to cellular energy metabolism. Mitochondrial RNA polymerase is thought to be the primase for mtDNA replication. However, it is unclear how this enzyme, which normally transcribes long polycistronic RNAs, can produce short RNA oligonucleotides to initiate mtDNA replication. We show that the PPR domain of Drosophila mitochondrial RNA polymerase (PolrMT) has 3'-to-5' exoribonuclease activity, which is indispensable for PolrMT to synthesize short RNA oligonucleotides and prime DNA replication in vitro. An exoribonuclease-deficient mutant, PolrMTE423P, partially restores mitochondrial transcription but fails to support mtDNA replication when expressed in PolrMT-mutant flies, indicating that the exoribonuclease activity is necessary for mtDNA replication. In addition, overexpression of PolrMTE423P in adult flies leads to severe neuromuscular defects and a marked increase in mtDNA transcript errors, suggesting that exoribonuclease activity may contribute to the proofreading of mtDNA transcription.

Higher levels of allograft injury in black patients early after heart transplantation.

Black patients suffer higher rates of antibody-mediated rejection and have worse long-term graft survival after heart transplantation. Donor-derived cell free DNA (ddcfDNA) is released into the blood following allograft injury. This study analyzed %ddcfDNA in 63 heart transplant recipients categorized by Black and non-Black race, during the first 200 days after transplant. Immediately after transplant, %ddcfDNA was higher for Black patients (mean [SE]: 8.3% [1.3%] vs 3.2% [1.2%], p = 0.001). In the first week post-transplant, the rate of decay in %ddcfDNA was similar (0.7% [0.68] vs 0.7% [0.11], p = 0.78), and values declined in both groups to a comparable plateau at 7 days post-transplant (0.46% [0.03] vs 0.45% [0.04], p = 0.78). The proportion of Black patients experiencing AMR was higher than non-Black patients (21% vs 9% [hazard ratio of 2.61 [95% confidence interval: 0.651-10.43], p = 0.18). Black patients were more likely to receive a race mismatched organ than non-Black patients (69% vs 35%, p = 0.01), which may explain the higher levels of early allograft injury.

Identification of Genes Contributing to a Long Circadian Period in Drosophila Melanogaster.

The endogenous circadian period of animals and humans is typically very close to 24 h. Individuals with much longer circadian periods have been observed, however, and in the case of humans, these deviations have health implications. Previously, we observed a line of Drosophila with a very long average period of 31.3 h for locomotor activity behavior. Preliminary mapping indicated that the long period did not map to known canonical clock genes but instead mapped to multiple chromosomes. Using RNA-Seq, we surveyed the whole transcriptome of fly heads from this line across time and compared it with a wild-type control. A three-way generalized linear model revealed that approximately two-thirds of the genes were expressed differentially among the two genotypes, while only one quarter of the genes varied across time. Using these results, we applied algorithms to search for genes that oscillated over 24 h, identifying genes not previously known to cycle. We identified 166 differentially expressed genes that overlapped with a previous Genome-wide Association Study (GWAS) of circadian behavior, strongly implicating them in the long-period phenotype. We tested mutations in 45 of these genes for their effect on the circadian period. Mutations in Alk, alph, CG10089, CG42540, CG6034, Kairos (CG6123), CG8768, klg, Lar, sick, and tinc had significant effects on the circadian period, with seven of these mutations increasing the circadian period of locomotor activity behavior. Genetic rescue of mutant Kairos restored the circadian period to wild-type levels, suggesting it has a critical role in determining period length in constant darkness.

Network Analysis and Transcriptome Profiling Identify Autophagic and Mitochondrial Dysfunctions in SARS-CoV-2 Infection.

Analyzing host cells' transcriptional response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection will help delineate biological processes underlying viral pathogenesis. First, analysis of expression profiles of lung cell lines A549 and Calu3 revealed upregulation of antiviral interferon signaling genes in response to all three SARS-CoV-2, MERS-CoV, or influenza A virus (IAV) infections. However, perturbations in expression of genes involved in inflammatory, mitochondrial, and autophagy processes were specifically observed in SARS-CoV-2-infected cells. Next, a validation study in infected human nasopharyngeal samples also revealed perturbations in autophagy and mitochondrial processes. Specifically, mTOR expression, mitochondrial ribosomal, mitochondrial complex I, lysosome acidification, and mitochondrial fission promoting genes were concurrently downregulated in both infected cell lines and human samples. SARS-CoV-2 infection impeded autophagic flux either by upregulating GSK3B in lung cell lines or by downregulating autophagy genes, SNAP29, and lysosome acidification genes in human samples, contributing to increased viral replication. Therefore, drugs targeting lysosome acidification or autophagic flux could be tested as intervention strategies. Finally, age-stratified SARS-CoV-2-positive human data revealed impaired upregulation of chemokines, interferon-stimulated genes, and tripartite motif genes that are critical for antiviral signaling. Together, this analysis has revealed specific aspects of autophagic and mitochondrial function that are uniquely perturbed in SARS-CoV-2-infected host cells.

Use of donor-derived-cell-free DNA as a marker of early allograft injury in primary graft dysfunction (PGD) to predict the risk of chronic lung allograft dysfunction (CLAD).

BACKGROUND: Primary graft dysfunction (PGD) is a risk factor for chronic lung allograft dysfunction (CLAD). However, the association between PGD and degree of allograft injury remains poorly defined. In this study, we leverage a novel biomarker for allograft injury, percentage donor-derived cell-free DNA (%ddcfDNA), to study the association between PGD, degree of allograft injury, and the development of CLAD. METHODS: This prospective cohort study recruited 99 lung transplant recipients and collected plasma samples on days 1, 3, and 7 for %ddcfDNA measurements. Clinical data on day 3 was used to adjudicate for PGD. %ddcfDNA levels were compared between PGD grades. In PGD patients, %ddcfDNA was compared between those who developed CLAD and those who did not. RESULTS: On posttransplant day 3, %ddcfDNA was higher in PGD than in non-PGD patients (median [IQR]: 12.2% [8.2, 22.0] vs 8.5% [5.6, 13.2] p = 0.01). %ddcfDNA correlated with the severity grade of PGD (r = 0.24, p = 0.02). Within the PGD group, higher levels of %ddcfDNA correlated with increased risk of developing CLAD (log OR(SE) 1.38 (0.53), p = 0.009). PGD patients who developed CLAD showed ∼2-times higher %ddcfDNA levels than patients who did not develop CLAD (median [IQR]: 22.4% [11.8, 27.6] vs 9.9% [6.7, 14.9], p = 0.007). CONCLUSION: PGD patients demonstrated increased early posttransplant allograft injury, as measured by %ddcfDNA, in comparison to non-PGD patients, and these high %ddcfDNA levels were associated with subsequent development of CLAD. This study suggests that %ddcfDNA identifies PGD patients at greater risk of CLAD than PGD alone.

Cell-free DNA maps COVID-19 tissue injury and risk of death and can cause tissue injury.

INTRODUCTIONThe clinical course of coronavirus 2019 (COVID-19) is heterogeneous, ranging from mild to severe multiorgan failure and death. In this study, we analyzed cell-free DNA (cfDNA) as a biomarker of injury to define the sources of tissue injury that contribute to such different trajectories.METHODSWe conducted a multicenter prospective cohort study to enroll patients with COVID-19 and collect plasma samples. Plasma cfDNA was subject to bisulfite sequencing. A library of tissue-specific DNA methylation signatures was used to analyze sequence reads to quantitate cfDNA from different tissue types. We then determined the correlation of tissue-specific cfDNA measures to COVID-19 outcomes. Similar analyses were performed for healthy controls and a comparator group of patients with respiratory syncytial virus and influenza.RESULTSWe found markedly elevated levels and divergent tissue sources of cfDNA in COVID-19 patients compared with patients who had influenza and/or respiratory syncytial virus and with healthy controls. The major sources of cfDNA in COVID-19 were hematopoietic cells, vascular endothelium, hepatocytes, adipocytes, kidney, heart, and lung. cfDNA levels positively correlated with COVID-19 disease severity, C-reactive protein, and D-dimer. cfDNA profile at admission identified patients who subsequently required intensive care or died during hospitalization. Furthermore, the increased cfDNA in COVID-19 patients generated excessive mitochondrial ROS (mtROS) in renal tubular cells in a concentration-dependent manner. This mtROS production was inhibited by a TLR9-specific antagonist.CONCLUSIONcfDNA maps tissue injury that predicts COVID-19 outcomes and may mechanistically propagate COVID-19-induced tissue injury.FUNDINGIntramural Targeted Anti-COVID-19 grant, NIH.

Donor-derived cell-free DNA accurately detects acute rejection in lung transplant patients, a multicenter cohort study.

BACKGROUND: Acute rejection, which includes antibody-mediated rejection and acute cellular rejection, is a risk factor for lung allograft loss. Lung transplant patients often undergo surveillance transbronchial biopsies to detect and treat acute rejection before irreversible chronic rejection develops. Limitations of this approach include its invasiveness and high interobserver variability. We tested the performance of percent donor-derived cell-free DNA (%ddcfDNA), a non-invasive blood test, to detect acute rejection. METHODS: This multicenter cohort study monitored 148 lung transplant subjects over a median of 19.6 months. We collected serial plasma samples contemporaneously with TBBx to measure %ddcfDNA. Clinical data was collected to adjudicate for acute rejection. The primary analysis consisted of computing the area-under-the-receiver-operating-characteristic-curve of %ddcfDNA to detect acute rejection. Secondary analysis determined %ddcfDNA rule-out thresholds for acute rejection. RESULTS: ddcfDNA levels were high after transplant surgery and decayed logarithmically. With acute rejection, ddcfDNA levels rose six-fold higher than controls. ddcfDNA levels also correlated with severity of lung function decline and histological grading of rejection. %ddcfDNA area-under-the-receiver-operating-characteristic-curve for acute rejection, AMR, and ACR were 0.89, 0.93, and 0.83, respectively. ddcfDNA levels of <0.5% and <1.0% showed a negative predictive value of 96% and 90% for acute rejection, respectively. Histopathology detected one-third of episodes with ddcfDNA levels ≥1.0%, even though >90% of these events were coincident to clinical complications missed by histopathology. CONCLUSIONS: This study demonstrates that %ddcfDNA reliably detects acute rejection and other clinical complications potentially missed by histopathology, lending support to its use as a non-invasive marker of allograft injury.

Network Analysis and Transcriptome Profiling Identify Autophagic and Mitochondrial Dysfunctions in SARS-CoV-2 Infection.

Analyzing host transcriptional changes in response to SARS-CoV-2 infection will help delineate biological processes underlying viral pathogenesis. Comparison of expression profiles of lung cell lines A549 (infected with either SARS-CoV-2 (with ACE2 expression)) or Influenza A virus (IAV)) and Calu3 (infected with SARS-CoV-2 or MERS-CoV) revealed upregulation of the antiviral interferon signaling in all three viral infections. However, perturbations in inflammatory, mitochondrial, and autophagy processes were specifically observed in SARS-CoV-2 infected cells. Validation of findings from cell line data revealed perturbations in autophagy and mitochondrial processes in the infected human nasopharyngeal samples. Specifically, downregulation of mTOR expression, mitochondrial ribosomal, mitochondrial complex I, and lysosome acidification genes were concurrently observed in both infected cell lines and human datasets. Furthermore, SARS-CoV-2 infection impedes autophagic flux by upregulating GSK3B in lung cell lines, or by downregulating autophagy genes, SNAP29 and lysosome acidification genes in human samples, contributing to increased viral replication. Therefore, drugs targeting lysosome acidification or autophagic flux could be tested as intervention strategies. Additionally, downregulation of MTFP1 (in cell lines) or SOCS6 (in human samples) results in hyperfused mitochondria and impede proper interferon response. Coexpression networks analysis identifies correlated clusters of genes annotated to inflammation and mitochondrial processes that are misregulated in SARS-CoV-2 infected cells. Finally, comparison of age stratified human gene expression data revealed impaired upregulation of chemokines, interferon stimulated and tripartite motif genes that are critical for antiviral signaling. Together, this analysis has revealed specific aspects of autophagic and mitochondrial function that are uniquely perturbed in SARS-CoV-2 infection.

Misregulation of ELK1, AP1, and E12 Transcription Factor Networks Is Associated with Melanoma Progression.

Melanoma is among the most malignant cutaneous cancers and when metastasized results in dramatically high mortality. Despite advances in high-throughput gene expression profiling in cancer transcriptomic studies, our understanding of mechanisms driving melanoma progression is still limited. We present here an in-depth bioinformatic analysis of the melanoma RNAseq, chromatin immunoprecipitation (ChIP)seq, and single-cell (sc)RNA seq data to understand cancer progression. Specifically, we have performed a consensus network analysis of RNA-seq data from clinically re-grouped melanoma samples to identify gene co-expression networks that are conserved in early (stage 1) and late (stage 4/invasive) stage melanoma. Overlaying the fold-change information on co-expression networks revealed several coordinately up or down-regulated subnetworks that may play a critical role in melanoma progression. Furthermore, by incorporating histone lysine-27 acetylation information and highly expressed genes identified from the single-cell RNA data from melanoma patient samples, we present a comprehensive list of pathways, putative protein-protein interactions (PPIs) and transcription factor (TF) networks that are driving cancer progression. From this analysis, we have identified Elk1, AP1 and E12 TF networks that coordinately change expression in late melanoma when compared to early melanoma, implicating these TFs in melanoma progression. Additionally, the sumoylation-associated interactome is upregulated in invasive melanoma. Together, this bioinformatic analysis potentially implicates a combination of TF networks and PPIs in melanoma progression, which if confirmed in the experimental systems, could be used as targets for drug intervention in melanoma.