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Platelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a noncanonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), DNA fibers released by neutrophils during inflammation. We investigated which host factors control NETs in vivo and found that two deoxyribonucleases (DNases), DNase1 and DNase1-like 3, degraded NETs in circulation during sterile neutrophilia and septicemia. In the absence of both DNases, intravascular NETs formed clots that obstructed blood vessels and caused organ damage. Vascular occlusions in patients with severe bacterial infections were associated with a defect to degrade NETs ex vivo and the formation of intravascular NET clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of intravascular NETs.

DNase1l3 is an endonuclease to degrade the chromatin of apoptotic or necrotic cells. Serum DNase1l3 may fulfill the function of clearance of chromatin released into the circulation by dying cells, which can trigger autoimmune responses. To date, it remains unclear whether serum DNase1l3 level associates with the pathogenesis of autoimmune diseases. Sixty-eight patients with dermatomyositis/polymyositis (DM/PM, n  = 30), systemic lupus erythematosus (SLE, n  = 20) and rheumatoid arthritis (RA, n  = 18), as well as 26 healthy blood donors were enrolled in the present study. Serum levels of DNase1l3 were quantified by enzyme-linked immunosorbent assay. DNASE1L3 activity in serum was estimated by the capability of serum to digest nucleosomal DNA. Clinical, biochemical, serological and other markers of disease activity (CRP, ESR, C3, C4, anti-Jo-1 and anti-dsDNA, etc.) were measured by standard laboratory procedure. We found a decrease in DNase1l3 level in the DM/PM and SLE patients, resulting in the reduction in serum activity to digest nucleosome DNA. In contrast, the level and activity of DNase1l3 remained unchanged in the RA patients. The DNase1l3 level was relatively lower in the DM/PM patients with anti-Jo-1 antibody and interstitial lung disease, and in the SLE patients with SLE disease activity index higher than 6, renal involvement and anti-dsDNA antibody. DNase1l3 level negatively correlated with CRP and IgG in the PM/DM patients and correlated with ESR in the SLE patients. We found a significant reduction in serum DNase1l3 level in DM/PM and SLE, which may associate with clinic features and disease activity.

In vitro selection experiments have produced nucleic acid ligands (aptamers) that bind tightly and specifically to a great variety of target biomolecules. The utility of aptamers is often limited by their vulnerability to nucleases present in biological materials. One way to circumvent this problem is to select an aptamer that binds the enantiomer of the target, then synthesize the enantiomer of the aptamer as a nuclease-insensitive ligand of the normal target. We have so identified a mirror-image single-stranded DNA that binds the peptide hormone vasopressin and have demonstrated its stability to nucleases and its bioactivity as a vasopressin antagonist in cell culture.

Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2 , associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans. Nucleic acid sensing is important to ensure that an innate immune response is only mounted against microbial nucleic acid. Here, the authors identify loss-of-function mutations in the DNASE2 gene that cause type I interferon-mediated autoinflammation due to enhanced systemic interferon signaling.

The CRISPR-Cas12a system has revolutionized nucleic acid testing (NAT) with its rapid and precise capabilities, yet it traditionally required RNA pre-amplification. Here we develop rapid fluorescence and lateral flow NAT assays utilizing a split Cas12a system (SCas12a), consisting of a Cas12a enzyme and a split crRNA. The SCas12a assay enables highly sensitive, amplification-free, and multiplexed detection of miRNAs and long RNAs without complex secondary structures. It can differentiate between mature miRNA and its precursor (pre-miRNA), a critical distinction for precise biomarker identification and cancer progression monitoring. The system’s specificity is further highlighted by its ability to detect DNA and miRNA point mutations. Notably, the SCas12a system can quantify the miR-21 biomarker in plasma from cervical cancer patients and, when combined with RPA, detect HPV at attomole levels in clinical samples. Together, our work presents a simple and cost-effective SCas12a-based NAT platform for various diagnostic settings. CRISPR-Cas12a nucleic acid detection largely involves pre-amplification and is limited in its ability to directly detect RNA. Here, authors present a split Cas12a system that enables direct, highly sensitive, and cost-effective detection of both DNA and RNA without amplification.

Circulating DNA in plasma consists of short DNA fragments. The biological processes generating such fragments are not well understood. DNASE1L3 is a secreted DNASE1-like nuclease capable of digesting DNA in chromatin, and its absence causes anti-DNA responses and autoimmunity in humans and mice. We found that the deletion of Dnase1l3 in mice resulted in aberrations in the fragmentation of plasma DNA. Such aberrations included an increase in short DNA molecules below 120 bp, which was positively correlated with anti-DNA antibody levels. We also observed an increase in long, multinucleosomal DNA molecules and decreased frequencies of the most common end motifs found in plasma DNA. These aberrations were independent of anti-DNA response, suggesting that they represented a primary effect of DNASE1L3 loss. Pregnant Dnase1l3 −/− mice carrying Dnase1l3 +/− fetuses showed a partial restoration of normal frequencies of plasma DNA end motifs, suggesting that DNASE1L3 from Dnase1l3-proficient fetuses could enter maternal systemic circulation and affect both fetal and maternal DNA fragmentation in a systemic as well as local manner. However, the observed shortening of circulating fetal DNA relative to maternal DNA was not affected by the deletion of Dnase1l3. Collectively, our findings demonstrate that DNASE1L3 plays a role in circulating plasma DNA homeostasis by enhancing fragmentation and influencing end-motif frequencies. These results support a distinct role of DNASE1L3 as a regulator of the physical form and availability of cell-free DNA and may have important implications for the mechanism whereby this enzyme prevents autoimmunity.

Endonuclease G and heart disease Elevated left ventricular mass, a highly heritable trait, is an important risk factor for heart failure and death. Stuart Cook and colleagues genetically dissect a locus in the rat associated with blood-pressure-independent cardiac hypertrophy and identify endonuclease G (ENDOG) as a key regulator of hypertrophy at this locus. They further show that the Endog gene is involved in proper mitochondrial function and is modulated by ERR-α and PGC1α, master regulators of mitochondrial and cardiac function. Loss of function of ENDOG causes impaired mitochondrial respiration and increased production of reactive oxygen species, which may contribute to the observed cardiac hypertrophy. Left ventricular mass (LVM) is a highly heritable trait 1 and an independent risk factor for all-cause mortality 2 . So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation 3 , and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood 4 , 5 . Unbiased systems genetics approaches in the rat 6 , 7 now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G ( Endog ), which previously was implicated in apoptosis 8 but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function) 9 , 10 , 11 , interaction of ENDOG with the mitochondrial genome and ENDOG -mediated regulation of mitochondrial mass. At baseline, the Endog -deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy.

This year's model The development of drugs for rheumatoid arthritis has not been helped by the lack of a good animal model. Now a newly developed mutant mouse system could help fill the gap. The mice lack the DNaseII gene, and are rescued from the lethal effects of that mutation by a second mutation that permits constitutive production of interferon. They develop chronic polyarthritis, resembling human rheumatoid arthritis, as a result of the failure of DNA degradation during apoptotic cell death and definitive erythropoiesis. This was unexpected: examination of various mouse tissues indicated that macrophages carrying undigested DNA become activated and produce TNF (tumour necrosis factor), leading to the development of polyarthritis. Interestingly TNF is involved in the pathogenesis of rheumatoid arthritis, and anti-TNF treatment is sometimes used to treat the disease. A large amount of chromosomal DNA is degraded during programmed cell death and definitive erythropoiesis 1 . DNase II is an enzyme that digests the chromosomal DNA of apoptotic cells and nuclei expelled from erythroid precursor cells after macrophages have engulfed them 1 , 2 . Here we show that DNase II -/- IFN-IR -/- mice and mice with an induced deletion of the DNase II gene develop a chronic polyarthritis resembling human rheumatoid arthritis. A set of cytokine genes was strongly activated in the affected joints of these mice, and their serum contained high levels of anti-cyclic citrullinated peptide antibody, rheumatoid factor and matrix metalloproteinase-3. Early in the pathogenesis, expression of the gene encoding tumour necrosis factor (TNF)-α was upregulated in the bone marrow, and administration of anti-TNF-α antibody prevented the development of arthritis. These results indicate that if macrophages cannot degrade mammalian DNA from erythroid precursors and apoptotic cells, they produce TNF-α, which activates synovial cells to produce various cytokines, leading to the development of chronic polyarthritis.

Besides genome editing, CRISPR-Cas12a has recently been used for DNA detection applications with attomolar sensitivity but, to our knowledge, it has not been used for the detection of small molecules. Bacterial allosteric transcription factors (aTFs) have evolved to sense and respond sensitively to a variety of small molecules to benefit bacterial survival. By combining the single-stranded DNA cleavage ability of CRISPR-Cas12a and the competitive binding activities of aTFs for small molecules and double-stranded DNA, here we develop a simple, supersensitive, fast and high-throughput platform for the detection of small molecules, designated CaT-SMelor ( C RISPR-Cas12a- and aT F-mediated s mall m ol e cu l e detect or ). CaT-SMelor is successfully evaluated by detecting nanomolar levels of various small molecules, including uric acid and p -hydroxybenzoic acid among their structurally similar analogues. We also demonstrate that our CaT-SMelor directly measured the uric acid concentration in clinical human blood samples, indicating a great potential of CaT-SMelor in the detection of small molecules. Bacterial allosteric transcription factors can sense and respond to a variety of small molecules. Here the authors present CaT-SMelor which uses Cas12a and allosteric transcription factors to detect small molecules in the nanomolar range.

The scarcity of malignant Hodgkin and Reed–Sternberg cells hampers tissue-based comprehensive genomic profiling of classic Hodgkin lymphoma (cHL). By contrast, liquid biopsies show promise for molecular profiling of cHL due to relatively high circulating tumour DNA (ctDNA) levels 1 – 4 . Here we show that the plasma representation of mutations exceeds the bulk tumour representation in most cases, making cHL particularly amenable to noninvasive profiling. Leveraging single-cell transcriptional profiles of cHL tumours, we demonstrate Hodgkin and Reed–Sternberg ctDNA shedding to be shaped by DNASE1L3, whose increased tumour microenvironment-derived expression drives high ctDNA concentrations. Using this insight, we comprehensively profile 366 patients, revealing two distinct cHL genomic subtypes with characteristic clinical and prognostic correlates, as well as distinct transcriptional and immunological profiles. Furthermore, we identify a novel class of truncating IL4R mutations that are dependent on IL-13 signalling and therapeutically targetable with IL-4Rα-blocking antibodies. Finally, using PhasED-seq 5 , we demonstrate the clinical value of pretreatment and on-treatment ctDNA levels for longitudinally refining cHL risk prediction and for detection of radiographically occult minimal residual disease. Collectively, these results support the utility of noninvasive strategies for genotyping and dynamic monitoring of cHL, as well as capturing molecularly distinct subtypes with diagnostic, prognostic and therapeutic potential. The potential use of circulating tumour DNA in classic Hodgkin lymphoma detection, classification and monitoring is defined.