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1.
Mol Biol Cell ; : mbcE22020066, 2022 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-35544303

RESUMO

Normal tissue and organ morphogenesis requires epithelial cell plasticity and conversion to a mesenchymal phenotype through a tightly regulated process: epithelial-to-mesenchymal transition (EMT). Alterations of EMT go far beyond cell-lineage segregation and contribute to pathologic conditions such as cancer. EMT is subject to intersecting control pathways; however, EMT's metabolic mechanism remains poorly understood. Here, we demonstrate that transforming growth factor ß (TGF-ß)-induced EMT is accompanied by decreased fatty acid oxidation (FAO) and reduced acetyl-coenzyme A (acetyl-CoA) levels. Acetyl-CoA is a central metabolite and the sole donor of acetyl groups to acetylate key proteins. Further, the short-chain fatty acid acetate increases acetyl-CoA levels-robustly inhibiting EMT and cancer cell migration. Acetate can restore EMT-associated α-tubulin acetylation levels, increasing microtubule stability. Transcriptome profiling and flow cytometric analysis show that acetate inhibits the global gene expression program associated with EMT and the EMT-associated G1 cell cycle arrest. Taken together, these results demonstrate that acetate is a potent metabolic regulator of EMT and that therapeutic manipulation of acetate metabolism could provide the basis for treating a wide range of EMT-linked pathological conditions, including cancer.

2.
Nat Cell Biol ; 24(5): 757-765, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35449456

RESUMO

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.

3.
Blood Adv ; 2022 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-35358998

RESUMO

In chronic lymphocytic leukemia (CLL), B-cell receptor signaling, tumor-microenvironment interactions and somatic mutations drive disease progression. To better understand the intersection between the microenvironment and molecular events in CLL pathogenesis, we integrated bulk transcriptome profiling of paired peripheral blood (PB) and lymph node (LN) samples from 34 patients. Oncogenic processes were upregulated in LN compared to PB, and in IGHV unmutated compared to mutated cases. Single-cell RNA sequencing distinguished 3 major cell states: quiescent, activated, and proliferating. The activated subpopulation comprised only 2.2% to 4.3% of the total tumor bulk in LN samples. RNA velocity analysis found that CLL cell fate in LN is unidirectional, starts in the proliferating state, transitions to the activated state, and ends in the quiescent state. A 10-gene signature derived from activated tumor cells was associated with inferior treatment-free survival and positively correlated with the proportion of activated CD4+ memory T cells and M2 macrophages in LN. Whole exome sequencing of paired PB and LN samples showed subclonal expansion in LN in approximately half of patients. Since mouse models have implicated activation-induced cytidine deaminase in mutagenesis, we compared AICDA expression between cases with and without clonal evolution, but did not find a difference. In contrast, the presence of a T-cell inflamed microenvironment in LN was associated with clonal stability. In summary, a distinct minor tumor subpopulation underlies CLL pathogenesis and drives clinical outcome. Clonal trajectories are shaped by the LN milieu where T-cell immunity may contribute to suppress clonal outgrowth. The clinical study is registered at clinicaltrials.gov as NCT00923507.

4.
Nat Neurosci ; 25(3): 381-389, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35260864

RESUMO

Recent genetic studies have identified variants associated with bipolar disorder (BD), but it remains unclear how brain gene expression is altered in BD and how genetic risk for BD may contribute to these alterations. Here, we obtained transcriptomes from subgenual anterior cingulate cortex and amygdala samples from post-mortem brains of individuals with BD and neurotypical controls, including 511 total samples from 295 unique donors. We examined differential gene expression between cases and controls and the transcriptional effects of BD-associated genetic variants. We found two coexpressed modules that were associated with transcriptional changes in BD: one enriched for immune and inflammatory genes and the other with genes related to the postsynaptic membrane. Over 50% of BD genome-wide significant loci contained significant expression quantitative trait loci (QTL) (eQTL), and these data converged on several individual genes, including SCN2A and GRIN2A. Thus, these data implicate specific genes and pathways that may contribute to the pathology of BP.


Assuntos
Transtorno Bipolar , Giro do Cíngulo , Tonsila do Cerebelo/metabolismo , Transtorno Bipolar/genética , Transtorno Bipolar/metabolismo , Encéfalo/metabolismo , Giro do Cíngulo/metabolismo , Humanos , Transcriptoma
5.
Blood Adv ; 2022 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-35271708

RESUMO

Acute pain, the most prominent complication of sickle cell disease (SCD), results from vaso-occlusion triggered by sickling of deoxygenated red blood cells (RBCs). Concentration of 2,3-diphosphoglycerate (2,3-DPG) in RBCs promotes deoxygenation by preferentially binding to the low-affinity T conformation of HbS. 2,3-DPG is an intermediate substrate in the glycolytic pathway in which pyruvate kinase (gene PKLR, protein PKR) is a rate-limiting enzyme; variants in PKLR may affect PKR activity, 2,3-DPG levels in RBCs, RBC sickling and acute pain episodes (APEs). We performed a candidate gene association study using 2 cohorts: 242 adult SCD-HbSS patients and 977 children with SCD-HbSS or SCD-HbSß0 thalassemia. Seven of 47 PKLR variants evaluated in the adult cohort were associated with hospitalization: intron 4 - rs2071053; intron 2 - rs8177970, rs116244351, rs114455416, rs12741350, rs3020781, and rs8177964. All 7 variants showed consistent effect directions in both cohorts and remained significant in weighted Fisher's meta-analyses of the adult and pediatric cohorts using p<0.0071 as threshold to correct for multiple testing. Allele-specific expression analyses in an independent cohort of 52 SCD adults showed that the intronic variants are likely to influence APE by affecting expression of PKLR, although the causal variant and mechanism are not defined.

6.
Res Sq ; 2022 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-35075453

RESUMO

COVID-19 pathogenesis is associated with an exuberant inflammatory response. However, the tissue injury pattern and immune response in solid-organ transplant recipients (SOTRs) taking immunosuppressive therapy have not been well characterized. Here, we perform both cfDNA and cytokine profiling on plasma samples to map tissue damage, including allograft injury and delineate underlying immunopathology. We identified injuries from multiple-tissue types, including hematopoietic cells, vascular endothelium, hepatocyte, adipocyte, pancreas, kidney, heart, and lung in SOTRs with COVID-19 that correlates with disease severity. SOTRs with COVID-19 have higher plasma levels of cytokines such as IFN-λ1, IFN-γ, IL-15, IL-18 IL-1RA, IL-6, MCP-2, and TNF-α as compared to healthy controls, and the levels of GM-CSF, IL-15, IL-6, IL-8, and IL-10 were associated with disease severity in SOTRs. Strikingly, IFN-λ and IP-10 were markedly increased in SOTRs compared to immunocompetent patients with COVID-19. Correlation analyses showed a strong association between monocyte-derived cfDNA and inflammatory cytokines/chemokines in SOTRs with COVID-19. Moreover, compared to other respiratory viral infections, COVID-19 induced pronounced injury in hematopoitic, vascular endothelial and endocrine tissues. Allograft injury, measured as donor-derived cfDNA was elevated in SOTRs with COVID-19, including allografts distant from the primary site of infection. Allograft injury correlated with inflammatory cytokines and cfDNA from immune cells. Furthermore, longitudinal analysis identified a gradual decrease of cfDNA and inflammatory cytokine levels in patients with a favorable outcome. Our findings highlight distinct tissue injury and cytokine features in SOTRs with COVID-19 that correlate with disease severity.

7.
J Clin Invest ; 132(5)2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-35025762

RESUMO

BACKGROUNDFasting and NAD+-boosting compounds, including NAD+ precursor nicotinamide riboside (NR), confer antiinflammatory effects. However, the underlying mechanisms and therapeutic potential are incompletely defined.METHODSWe explored the underlying biology in myeloid cells from healthy volunteers following in vivo placebo or NR administration and subsequently tested the findings in vitro in monocytes extracted from patients with systemic lupus erythematosus (SLE).RESULTSRNA-Seq of unstimulated and LPS-activated monocytes implicated NR in the regulation of autophagy and type I IFN signaling. In primary monocytes, NR blunted LPS-induced IFN-ß production, and genetic or pharmacological disruption of autophagy phenocopied this effect. Given that NAD+ is a coenzyme in oxidoreductive reactions, metabolomics was performed and identified that NR increased the inosine level. Inosine supplementation similarly blunted autophagy and IFN-ß release. Finally, because SLE exhibits type I IFN dysregulation, we assessed the NR effect on monocytes from patients with SLE and found that NR reduced autophagy and IFN-ß release.CONCLUSIONWe conclude that NR, in an NAD+-dependent manner and in part via inosine signaling, mediated suppression of autophagy and attenuated type I IFN in myeloid cells, and we identified NR as a potential adjunct for SLE management.TRIAL REGISTRATIONClinicalTrials.gov registration numbers NCT02812238, NCT00001846, and NCT00001372.FUNDINGThis work was supported by the NHLBI and NIAMS Intramural Research divisions.


Assuntos
Lúpus Eritematoso Sistêmico , NAD , Estudos Clínicos como Assunto , Humanos , Inosina , Interferon beta , Lipopolissacarídeos , Monócitos , Niacinamida , Receptor 4 Toll-Like
8.
J Physiol ; 600(3): 547-567, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34837710

RESUMO

Mitochondrial adaptations are fundamental to differentiated function and energetic homeostasis in mammalian cells. But the mechanisms that underlie these relationships remain poorly understood. Here, we investigated organ-specific mitochondrial morphology, connectivity and protein composition in a model of extreme mammalian metabolism, the least shrew (Cryptotis parva). This was achieved through a combination of high-resolution 3D focused ion beam electron microscopy imaging and tandem mass tag mass spectrometry proteomics. We demonstrate that liver and kidney mitochondrial content are equivalent to the heart, permitting assessment of mitochondrial adaptations in different organs with similar metabolic demand. Muscle mitochondrial networks (cardiac and skeletal) are extensive, with a high incidence of nanotunnels - which collectively support the metabolism of large muscle cells. Mitochondrial networks were not detected in the liver and kidney as individual mitochondria are localized with sites of ATP consumption. This configuration is not observed in striated muscle, likely due to a homogeneous ATPase distribution and the structural requirements of contraction. These results demonstrate distinct, fundamental mitochondrial structural adaptations for similar metabolic demand that are dependent on the topology of energy utilization process in a mammalian model of extreme metabolism. KEY POINTS: Least shrews were studied to explore the relationship between metabolic function, mitochondrial morphology and protein content in different tissues. Liver and kidney mitochondrial content and enzymatic activity approaches that of the heart, indicating similar metabolic demand among tissues that contribute to basal and maximum metabolism. This allows an examination of mitochondrial structure and composition in tissues with similar maximum metabolic demands. Mitochondrial networks only occur in striated muscle. In contrast, the liver and kidney maintain individual mitochondria with limited reticulation. Muscle mitochondrial reticulation is the result of dense ATPase activity and cell-spanning myofibrils which require networking for adequate metabolic support. In contrast, liver and kidney ATPase activity is localized to the endoplasmic reticulum and basolateral membrane, respectively, generating a locally balanced energy conversion and utilization. Mitochondrial morphology is not driven by maximum metabolic demand, but by the cytosolic distribution of energy-utilizing systems set by the functions of the tissue.


Assuntos
Músculo Estriado , Musaranhos , Animais , Metabolismo Energético/fisiologia , Mitocôndrias/metabolismo , Músculo Esquelético/fisiologia , América do Norte , Musaranhos/anatomia & histologia
9.
Genome Biol ; 23(1): 2, 2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34980216

RESUMO

BACKGROUND: Reproducible detection of inherited variants with whole genome sequencing (WGS) is vital for the implementation of precision medicine and is a complicated process in which each step affects variant call quality. Systematically assessing reproducibility of inherited variants with WGS and impact of each step in the process is needed for understanding and improving quality of inherited variants from WGS. RESULTS: To dissect the impact of factors involved in detection of inherited variants with WGS, we sequence triplicates of eight DNA samples representing two populations on three short-read sequencing platforms using three library kits in six labs and call variants with 56 combinations of aligners and callers. We find that bioinformatics pipelines (callers and aligners) have a larger impact on variant reproducibility than WGS platform or library preparation. Single-nucleotide variants (SNVs), particularly outside difficult-to-map regions, are more reproducible than small insertions and deletions (indels), which are least reproducible when > 5 bp. Increasing sequencing coverage improves indel reproducibility but has limited impact on SNVs above 30×. CONCLUSIONS: Our findings highlight sources of variability in variant detection and the need for improvement of bioinformatics pipelines in the era of precision medicine with WGS.


Assuntos
Genoma Humano , Polimorfismo de Nucleotídeo Único , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL , Reprodutibilidade dos Testes , Sequenciamento Completo do Genoma
10.
Front Immunol ; 12: 757279, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34917079

RESUMO

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a widely available curative option for patients with sickle cell disease (SCD). Our original non-myeloablative haplo-HSCT trial employing post-transplant (PT) cyclophosphamide had a low incidence of GVHD but had high rejection rates. Here, we aimed to evaluate immune reconstitution following haplo-HSCT and identify cytokines and cells associated with graft rejection/engraftment. 50 cytokines and 10 immune cell subsets were screened using multiplex-ELISA and flow cytometry, respectively, at baseline and PT-Days 30, 60, 100, and 180. We observed the most significant differences in cytokine levels between the engrafted and rejected groups at PT-Day 60, corresponding with clinical findings of secondary graft rejection. Of the 44 cytokines evaluated, plasma concentrations of 19 cytokines were different between the two groups at PT-Day 60. Factor analysis suggested two independent factors. The first factor (IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, and TGFb1 contributed significantly) was strongly associated with engraftment with OR = 2.7 (95%CI of 1.4 to 5.4), whereas the second factor (GROa and IL-18 contributed significantly) was not significantly associated with engraftment. Sufficient donor myeloid chimerism (DMC) is critical for the success of HSCT; here, we evaluated immune cells among high (H) DMC (DMC≥20%) and low (L) DMC (DMC<20%) groups along with engrafted and rejected groups. We found that early myeloid-derived suppressor cell (eMDSC) frequencies were elevated in engrafted patients and patients with HDMC at PT-Day 30 (P< 0.04 & P< 0.003, respectively). 9 of 20 patients were evaluated for the source of eMDSCs. The HDMC group had high mixed chimeric eMDSCs as compared to the LDMC group (P< 0.00001). We found a positive correlation between the frequencies of eMDSCs and Tregs at PT-Day 100 (r=0.72, P <0.0007); eMDSCs at BSL and Tregs at PT-Day 100 (r=0.63, P <0.004). Of 10 immune regulatory cells and 50 cytokines, we observed mixed chimeric eMDSCs and IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, TGFb1 as potential hits which could serve as prognostic markers in predicting allograft outcome towards engraftment following haploidentical HSCT employing post-transplant cyclophosphamide. The current findings need to be replicated and further explored in a larger cohort.


Assuntos
Anemia Falciforme/terapia , Transplante de Células-Tronco Hematopoéticas , Reconstituição Imune/imunologia , Quimeras de Transplante , Adulto , Anemia Falciforme/imunologia , Quimerismo , Ciclofosfamida/uso terapêutico , Citocinas/imunologia , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Humanos , Imunossupressores/uso terapêutico , Células Supressoras Mieloides , Prognóstico , Condicionamento Pré-Transplante , Transplante Haploidêntico , Resultado do Tratamento
11.
Cardiovasc Res ; 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34668514

RESUMO

AIMS: Prolyl hydroxylation is a post-translational modification that regulates protein stability, turnover, and activity. The proteins that catalyze prolyl hydroxylation belong to the 2-oxoglutarate- and iron-dependent oxygenase family of proteins. 2-oxoglutarate- and iron-dependent oxygenase domain-containing protein 1 (Ogfod1), which hydroxylates a proline in ribosomal protein s23 is a newly-described member of this family. The aims of this study were to investigate roles for Ogfod1 in the heart, and in the heart's response to stress. METHODS AND RESULTS: We isolated hearts from wild type (WT) and Ogfod1 knockout (KO) mice and performed quantitative proteomics using Tandem Mass Tag labelling coupled to Liquid Chromatography and tandem Mass Spectrometry (LC-MS/MS) to identify protein changes. Ingenuity Pathway Analysis identified "Urate Biosynthesis/Inosine 5'-phosphate Degradation" and "Purine Nucleotides Degradation II (Aerobic)" as the most significantly-enriched pathways. We performed metabolomics analysis and found that both purine and pyrimidine pathways were altered with the purine nucleotide inosine 5'-monophosphate (IMP) showing a 3.5-fold enrichment in KO hearts (P = 0.011) and the pyrimidine catabolism product beta-alanine showing a 1.7-fold enrichment in KO hearts (P = 0.014). As changes in these pathways have been shown to contribute to cardioprotection, we subjected isolated perfused hearts to ischemia and reperfusion (I/R). KO hearts showed a 41.4% decrease in infarct size and a 34% improvement in cardiac function compared to WT hearts. This protection was also evident in an in vivo I/R model. Additionally, our data show that treating isolated perfused WT hearts with carnosine, a metabolite of beta-alanine, improved protection in the context of I/R injury, whereas treating KO hearts with carnosine had no impact on recovery of function or infarct size. CONCLUSIONS: Taken together, these data show that Ogfod1 deletion alters the myocardial proteome and metabolome to confer protection against I/R injury. TRANSLATIONAL PERSPECTIVE: Heart disease is the leading cause of death in the US. In characterizing the cardiovascular effects of deleting the prolyl hydroxylase Ogfod1 and investigating its role in disease pathology, we found that deleting Ogfod1 protected hearts against ex vivo and in vivo I/R injury. Ogfod1-KO hearts showed significant metabolomic and proteomic changes that supported altered purine and pyrimidine nucleotide synthesis and turnover. Beta-alanine, a precursor of the anti-oxidant carnosine and a product of pyrimidine degradation, accumulated in KO hearts to help confer cardioprotection. Altogether, these data suggest a role for Ogfod1 downregulation as a therapeutic strategy for heart disease.

13.
Stem Cell Reports ; 16(9): 2336-2350, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34450041

RESUMO

Activation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs.


Assuntos
Hipóxia Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Receptores Notch/metabolismo , Antígenos CD34/metabolismo , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Biologia Computacional/métodos , Humanos , Anotação de Sequência Molecular , Receptores Notch/genética , Transdução de Sinais , Transcriptoma
14.
Nutrients ; 13(5)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33924911

RESUMO

Intermittent fasting and fasting mimetic diets ameliorate inflammation. Similarly, serum extracted from fasted healthy and asthmatic subjects' blunt inflammation in vitro, implicating serum components in this immunomodulation. To identify the proteins orchestrating these effects, SOMAScan technology was employed to evaluate serum protein levels in healthy subjects following an overnight, 24-h fast and 3 h after refeeding. Partial least square discriminant analysis identified several serum proteins as potential candidates to confer feeding status immunomodulation. The characterization of recombinant IGFBP1 (elevated following 24 h of fasting) and PYY (elevated following refeeding) in primary human CD4+ T cells found that they blunted and induced immune activation, respectively. Furthermore, integrated univariate serum protein analysis compared to RNA-seq analysis from peripheral blood mononuclear cells identified the induction of IL1RL1 and MFGE8 levels in refeeding compared to the 24-h fasting in the same study. Subsequent quantitation of these candidate proteins in lean versus obese individuals identified an inverse regulation of serum levels in the fasted subjects compared to the obese subjects. In parallel, IL1RL1 and MFGE8 supplementation promoted increased CD4+ T responsiveness to T cell receptor activation. Together, these data show that caloric load-linked conditions evoke serological protein changes, which in turn confer biological effects on circulating CD4+ T cell immune responsiveness.


Assuntos
Proteínas Sanguíneas/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Jejum/metabolismo , Inflamação/sangue , Nutrientes/sangue , Obesidade/sangue , Adulto , Idoso , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Reprodutibilidade dos Testes , Adulto Jovem
15.
Genome Biol ; 22(1): 111, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863366

RESUMO

BACKGROUND: Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. RESULTS: In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5-100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. CONCLUSION: These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.


Assuntos
Alelos , Biomarcadores Tumorais , Frequência do Gene , Testes Genéticos/métodos , Variação Genética , Genômica/métodos , Neoplasias/genética , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Heterogeneidade Genética , Testes Genéticos/normas , Genômica/normas , Humanos , Neoplasias/diagnóstico , Fluxo de Trabalho
16.
Front Genet ; 12: 599261, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33796130

RESUMO

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.

17.
Blood ; 137(22): 3116-3126, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33661274

RESUMO

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.


Assuntos
Anemia Falciforme/sangue , Ácidos Nucleicos Livres/sangue , Metilação de DNA , DNA Mitocondrial/sangue , Adulto , Idoso , Biomarcadores/sangue , Armadilhas Extracelulares/metabolismo , Feminino , Humanos , Inflamação/sangue , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Nucleotidiltransferases/metabolismo , Transdução de Sinais
18.
JCI Insight ; 6(7)2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33651717

RESUMO

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.


Assuntos
COVID-19 , Ácidos Nucleicos Livres , Insuficiência de Múltiplos Órgãos , Especificidade de Órgãos/genética , SARS-CoV-2 , Biomarcadores/análise , Biomarcadores/sangue , COVID-19/sangue , COVID-19/complicações , COVID-19/diagnóstico , COVID-19/mortalidade , Ácidos Nucleicos Livres/análise , Ácidos Nucleicos Livres/sangue , Estudos de Coortes , Metilação de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência de Múltiplos Órgãos/sangue , Insuficiência de Múltiplos Órgãos/diagnóstico , Insuficiência de Múltiplos Órgãos/etiologia , Avaliação de Resultados em Cuidados de Saúde , Prognóstico , Estudos Prospectivos , Reprodutibilidade dos Testes , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/patogenicidade , Índice de Gravidade de Doença , Estados Unidos/epidemiologia
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