Protection of ß2GPI Deficient Mice from Thrombosis Reflects a Defect in PAR3-facilitated Platelet Activation.

Background: Antibodies to ß2-glycoprotein I (ß2GPI) cause thrombosis in antiphospholipid syndrome, however the role of ß2GPI itself in regulation of coagulation pathways in vivo is not well understood. Methods: We developed ß2GPI-deficient mice (Apoh -/- ) by deleting exon 2 and 3 of Apoh using CRISPR/Cas9 and compared the propensity of wild-type (WT) and Apoh -/- mice to develop thrombosis using rose bengal and FeCl 3 -induced carotid thrombosis, laser-induced cremaster arteriolar injury, and inferior vena cava (IVC) stasis models. We also compared tail bleeding times and assessed platelet activation in WT and Apoh -/- mice in the absence and presence of exogenous ß2GPI. Results: Compared to WT littermates, Apoh -/- mice demonstrated a prolonged time to occlusion of the carotid artery after exposure to rose bengal or FeCl 3 , and reduced platelet and fibrin accumulation in cremasteric arterioles after laser injury. Similarly, significantly smaller thrombi were retrieved from the IVC of Apoh -/- mice 48 hours after IVC occlusion. The activated partial thromboplastin time (aPTT) and prothrombin time, as well as aPTT reagent- and tissue factor-induced thrombin generation times using plasma from Apoh -/- and WT mice revealed no differences. However, we observed significant prolongation of tail bleeding in Apoh -/- mice, and reduced P-selectin expression and binding of fibrinogen to the activated α2bß3 integrin on platelets from these mice after stimulation with low thrombin concentrations; these changes were reversed by exogenous ß2GPI. An antibody to PAR3 blocked thrombin-induced activation of WT, but not Apoh -/- platelets, as well as the ability of ß2GPI to restore the activation response of Apoh -/- platelets to thrombin. ß2GPI deficiency did not affect platelet activation by a PAR4-activator peptide, or ADP. Conclusions: In mice, ß2GPI may mediate procoagulant activity by enhancing the ability of PAR3 to present thrombin to PAR4, promoting platelet activation at low thrombin concentrations. Key Points: ß2GPI deficient mice are protected from experimental arterial, venous, and microvascular thrombosis.ß2GPI deficient mice display prolonged tail bleeding times and reduced PAR3-facilitated platelet activation by low concentrations of thrombin.

LCK facilitates DNA damage repair by stabilizing RAD51 and BRCA1 in the nucleus of chemoresistant ovarian cancer.

Poly-ADP Ribose Polymerase (PARP) targeted therapy is clinically approved for the treatment of homologous recombination (HR) repair deficient tumors. The remarkable success of this therapy in the treatment of HR repair deficient cancers has not translated to HR-proficient cancers. Our studies identify the novel role of non-receptor lymphocyte-specific protein tyrosine kinase (LCK) in the regulation of HR repair in endometrioid epithelial ovarian cancer (eEOC) model. We show that DNA damage leads to direct interaction of LCK with the HR repair proteins RAD51 and BRCA1 in a kinase dependent manner RAD51 and BRCA1 stabilization. LCK expression is induced and activated in the nucleus in response to DNA damage insult. Disruption of LCK expression attenuates RAD51, BRCA1, and BRCA2 protein expression by hampering there stability and results in inhibition of HR-mediated DNA repair including suppression of RAD51 foci formation, and augmentation of γH2AX foci formation. In contrast LCK overexpression leads to increased RAD51 and BRCA1 expression with a concomitant increase in HR DNA damage repair. Importantly, attenuation of LCK sensitizes HR-proficient eEOC cells to PARP inhibitor in cells and pre-clinical mouse studies. Collectively, our findings identify a novel therapeutic strategy to expand the utility of PARP targeted therapy in HR proficient ovarian cancer.

Polyphosphate expression by cancer cell extracellular vesicles mediates binding of factor XII and contact activation.

Extracellular vesicles (EV) have been implicated in diverse biological processes, including intracellular communication, transport of nucleic acids, and regulation of vascular function. Levels of EVs are elevated in cancer, and studies suggest that EV may stimulate thrombosis in patients with cancer through expression of tissue factor. However, limited data also implicate EV in the activation of the contact pathway of coagulation through activation of factor XII (FXII) to FXIIa. To better define the ability of EV to initiate contact activation, we compared the ability of EV derived from different cancer cell lines to activate FXII. EV from all cell lines activated FXII, with those derived from pancreatic and lung cancer cell lines demonstrating the most potent activity. Concordant with the activation of FXII, EV induced the cleavage of high molecular weight kininogen (HK) to cleaved kininogen. We also observed that EVs from patients with cancer stimulated FXII activation and HK cleavage. To define the mechanisms of FXII activation by EV, EV were treated with calf intestinal alkaline phosphatase or Escherichia coli exopolyphosphatase to degrade polyphosphate; this treatment blocked binding of FXII to EVs and the ability of EV to mediate FXII activation. In vivo, EV induced pulmonary thrombosis in wild-type mice, with protection conferred by a deficiency in FXII, HK, or prekallikrein. Moreover, pretreatment of EVs with calf intestinal alkaline phosphatase inhibited their prothrombotic effect. These results indicate that polyphosphate mediates the binding of contact factors to EV and that EV-associated polyphosphate may contribute to the prothrombotic effects of EV in cancer.

Stress Granule-Mediated Oxidized RNA Decay in P-Body: Hypothetical Role of ADAR1, Tudor-SN, and STAU1.

Reactive oxygen species (ROS) generated under oxidative stress (OS) cause oxidative damage to RNA. Recent studies have suggested a role for oxidized RNA in several human disorders. Under the conditions of oxidative stress, mRNAs released from polysome dissociation accumulate and initiate stress granule (SG) assembly. SGs are highly enriched in mRNAs, containing inverted repeat (IR) Alus in 3' UTRs, AU-rich elements, and RNA-binding proteins. SGs and processing bodies (P-bodies) transiently interact through a docking mechanism to allow the exchange of RNA species. However, the types of RNA species exchanged, and the mechanisms and outcomes of exchange are still unknown. Specialized RNA-binding proteins, including adenosine deaminase acting on RNA (ADAR1-p150), with an affinity toward inverted repeat Alus, and Tudor staphylococcal nuclease (Tudor-SN) are specifically recruited to SGs under OS along with an RNA transport protein, Staufen1 (STAU1), but their precise biochemical roles in SGs and SG/P-body docking are uncertain. Here, we critically review relevant literature and propose a hypothetical mechanism for the processing and decay of oxidized-RNA in SGs/P-bodies, as well as the role of ADAR1-p150, Tudor-SN, and STAU1.

The J-elongated conformation of ß2-glycoprotein I predominates in solution: implications for our understanding of antiphospholipid syndrome.

ß2-Glycoprotein I (ß2GPI) is an abundant plasma protein displaying phospholipid-binding properties. Because it binds phospholipids, it is a target of antiphospholipid antibodies (aPLs) in antiphospholipid syndrome (APS), a life-threatening autoimmune thrombotic disease. Indeed, aPLs prefer membrane-bound ß2GPI to that in solution. ß2GPI exists in two almost equally populated redox states: oxidized, in which all the disulfide bonds are formed, and reduced, in which one or more disulfide bonds are broken. Furthermore, ß2GPI can adopt multiple conformations (i.e. J-elongated, S-twisted, and O-circular). While strong evidence indicates that the J-form is the structure bound to aPLs, which conformation exists and predominates in solution remains controversial, and so is the conformational pathway leading to the bound state. Here, we report that human recombinant ß2GPI purified under native conditions is oxidized. Moreover, under physiological pH and salt concentrations, this oxidized form adopts a J-elongated, flexible conformation, not circular or twisted, in which the N-terminal domain I (DI) and the C-terminal domain V (DV) are exposed to the solvent. Consistent with this model, binding kinetics and mutagenesis experiments revealed that in solution the J-form interacts with negatively charged liposomes and with MBB2, a monoclonal anti-DI antibody that recapitulates most of the features of pathogenic aPLs. We conclude that the preferential binding of aPLs to phospholipid-bound ß2GPI arises from the ability of its preexisting J-form to accumulate on the membranes, thereby offering an ideal environment for aPL binding. We propose that targeting the J-form of ß2GPI provides a strategy to block pathogenic aPLs in APS.

Complement activity and complement regulatory gene mutations are associated with thrombosis in APS and CAPS.

The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies, including anti-ß2-glycoprotein-I (anti-ß2GPI), that are considered central to APS pathogenesis. Based on animal studies showing a role of complement in APS-related clinical events, we used the modified Ham (mHam) assay (complement-dependent cell killing) and cell-surface deposition of C5b-9 to test the hypothesis that complement activation is associated with thrombotic events in APS. A positive mHam (and corresponding C5b-9 deposition) were present in 85.7% of catastrophic APS (CAPS), 35.6% of APS (and 68.5% of samples collected within 1 year of thrombosis), and only 6.8% of systemic lupus erythematosus (SLE) sera. A positive mHam assay was associated with triple positivity (for lupus anticoagulant, anticardiolipin, and anti-ß2GPI antibodies) and recurrent thrombosis. Patient-derived anti-ß2GPI antibodies also induced C5b-9 deposition, which was blocked completely by an anti-C5 monoclonal antibody, but not by a factor D inhibitor, indicating that complement activation by anti-ß2GPI antibodies occurs primarily through the classical complement pathway. Finally, patients with CAPS have high rates of rare germline variants in complement regulatory genes (60%), compared with patients with APS (21.8%) or SLE (28.6%) or normal controls (23.3%), and have mutations at a rate similar to that of patients with atypical hemolytic uremic syndrome (51.5%). Taken together, our data suggest that anti-ß2GPI antibodies activate complement and contribute to thrombosis in APS, whereas patients with CAPS have underlying mutations in complement regulatory genes that serve as a "second hit," leading to uncontrolled complement activation and a more severe thrombotic phenotype.

Extracellular Vesicles in the Antiphospholipid Syndrome.

Antiphospholipid antibodies (aPL), particularly those directed against ß2-glycoprotein I, cause activation of vascular cells (endothelial cells, platelets, monocytes) and release of extracellular vesicles (EVs), which include exosomes and microparticles (MPs). MPs, particularly endothelial MPs, have been most extensively studied in antiphospholipid syndrome (APS). Compared with healthy controls, patients with aPL have significantly higher levels of circulating endothelial and platelet MPs, including MPs expressing immunological and functional tissue factor. Although a consistent relationship of EVs with APS-related thrombosis and obstetric events has not yet been demonstrated, elevated levels of MPs occurring remote from thrombotic events suggest a chronic state of vascular activation in APS. In addition to being a marker of cellular activation, EVs express bioactive lipids, proteins, and nucleic acids, particularly microribonucleic acid (microRNA). EVs may potentially play a pathogenic role in APS by stimulating thrombosis through tissue factor-dependent and independent mechanisms and by promoting vascular activation. Further research is needed to understand these mechanisms and to determine whether EVs may be a useful biomarker to identify patients with aPL at highest risk of clinical events.

CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors.

Effective targeting of cancer stem cells (CSCs) requires neutralization of self-renewal and chemoresistance, but these phenotypes are often regulated by distinct molecular mechanisms. Here we report the ability to target both of these phenotypes via CD55, an intrinsic cell surface complement inhibitor, which was identified in a comparative analysis between CSCs and non-CSCs in endometrioid cancer models. In this context, CD55 functions in a complement-independent manner and required lipid raft localization for CSC maintenance and cisplatin resistance. CD55 regulated self-renewal and core pluripotency genes via ROR2/JNK signaling and in parallel cisplatin resistance via lymphocyte-specific protein tyrosine kinase (LCK) signaling, which induced DNA repair genes. Targeting LCK signaling via saracatinib, an inhibitor currently undergoing clinical evaluation, sensitized chemoresistant cells to cisplatin. Collectively, our findings identify CD55 as a unique signaling node that drives self-renewal and therapeutic resistance through a bifurcating signaling axis and provides an opportunity to target both signaling pathways in endometrioid tumors.

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies. Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important. The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

Novel role for p56/Lck in regulation of endothelial cell survival and angiogenesis.

Previous studies have demonstrated that cleaved high-molecular-weight kininogen (HKa) induces endothelial apoptosis and inhibits angiogenesis and have suggested that this occurs through inhibition of Src family kinases. This study assessed the role of tyrosine-protein kinase Lck (p56/Lck) in this pathway. We analyzed early events leading to apoptosis of human endothelial cells exposed to HKa. The role of p56/Lck was investigated using short interfering (si) RNA knockdown and lentivirus expression in assays of endothelial tube formation, sprouting of neovessels from murine aorta, and angiogenesis in Matrigel plugs. HKa stimulated expression and phosphorylation of p56/Lck. siRNA knockdown of p56/Lck promoted endothelial proliferation and blocked HKa-induced apoptosis and activation of p53, Bax, and Bak. Lentivirus expression of p56/Lck in endothelial cells induced apoptosis and blocked tube formation. Expression of p56/Lck in murine aortic rings blocked sprouting angiogenesis. Lentivirus expressing p56/Lck blocked angiogenesis in Matrigel plugs, while p56/Lck short hairpin RNA inhibited the antiangiogenic effect of HKa. Scrambled siRNAs and empty lentiviral vectors were used in all experiments. Apoptosis of proliferating endothelial cells and inhibition of angiogenesis by HKa requires p56/Lck. This suggests a novel role for p56/Lck in regulation of endothelial cell survival and angiogenesis.-Betapudi, V., Shukla, M., Alluri, R., Merkulov, S., McCrae, K. R. Novel role for p56/Lck in regulation of endothelial cell survival and angiogenesis.

Halothane modulates the type i interferon response to influenza and minimizes the risk of secondary bacterial pneumonia through maintenance of neutrophil recruitment in an animal model.

BACKGROUND: To minimize the risk of pneumonia, many anesthesiologists delay anesthesia-requiring procedures when patients exhibit signs of viral upper respiratory tract infection. Postinfluenza secondary bacterial pneumonias (SBPs) are a major cause of morbidity and mortality. An increased host susceptibility to SBP postinfluenza has been attributed to physical damage to the pulmonary epithelium, but flu-induced effects on the immune system are being shown to also play an important role. The authors demonstrate that halothane mitigates the risk of SBP postflu through modulation of the effects of type I interferon (IFN). METHODS: Mice (n = 6 to 15) were exposed to halothane or ketamine and treated with influenza and Streptococcus pneumoniae. Bronchoalveolar lavage and lung homogenate were procured for the measurement of inflammatory cells, cytokines, chemokines, albumin, myeloperoxidase, and bacterial load. RESULTS: Halothane exposure resulted in decreased bacterial burden (7.9 ± 3.9 × 10 vs. 3.4 ± 1.6 × 10 colony-forming units, P < 0.01), clinical score (0.6 ± 0.2 vs. 2.3 ± 0.2, P < 0.0001), and lung injury (as measured by bronchoalveolar lavage albumin, 1.5 ± 0.7 vs. 6.8 ± 1.6 mg/ml, P < 0.01) in CD-1 mice infected with flu for 7 days and challenged with S. pneumoniae on day 6 postflu. IFN receptor A1 knockout mice similarly infected with flu and S. pneumoniae, but not exposed to halothane, demonstrated a reduction of lung bacterial burden equivalent to that achieved in halothane-exposed wild-type mice. CONCLUSION: These findings indicate that the use of halogenated volatile anesthetics modulates the type I IFN response to influenza and enhance postinfection antibacterial immunity.

Pathophysiological role of microRNA-29 in pancreatic cancer stroma.

Dense fibrotic stroma associated with pancreatic ductal adenocarcinoma (PDAC) is a major obstacle for drug delivery to the tumor bed and plays a crucial role in pancreatic cancer progression. Current, anti-stromal therapies have failed to improve tumor response to chemotherapy and patient survival. Furthermore, recent studies show that stroma impedes tumor progression, and its complete ablation accelerates PDAC progression. In an effort to understand the molecular mechanisms associated with tumor-stromal interactions, using in vitro and in vivo models and PDAC patient biopsies, we show that the loss of miR-29 is a common phenomenon of activated pancreatic stellate cells (PSCs)/fibroblasts, the major stromal cells responsible for fibrotic stromal reaction. Loss of miR-29 is correlated with a significant increase in extracellular matrix (ECM) deposition, a major component in PDAC stroma. Our in vitro miR-29 gain/loss-of-function studies document the role of miR-29 in PSC-mediated ECM stromal protein accumulation. Overexpression of miR-29 in activated stellate cells reduced stromal deposition, cancer cell viability, and cancer growth in co-culture. Furthermore, the loss of miR-29 in TGF-ß1 activated PSCs is SMAD3 dependent. These results provide insights into the mechanistic role of miR-29 in PDAC stroma and its potential use as a therapeutic agent to target PDAC.

Successful surgical outcome of infected aneurysmal drug-coated stent endocarditis.

A 52-year-old man developed pyrexia after primary angioplasty following anterior myocardial infarction Noninvasive evaluation revealed staphylococcal septicemia with suspicion of an aneurysm or abscess at the stented segment. Coronary angiography confirmed the presence of a large aneurysm. The infected left anterior descending artery aneurysm was de-roofed and ligated, and a saphenous vein bypass graft was placed, with an excellent outcome.

Low pH environmental stress inhibits LPS and LTA-stimulated proinflammatory cytokine production in rat alveolar macrophages.

Gastric aspiration increases the risks for developing secondary bacterial pneumonia. Cytokine elaboration through pathogen recognition receptors (PRRs) is an important mechanism in initiating innate immune host response. Effects of low pH stress, a critical component of aspiration pathogenesis, on the PRR pathways were examined, specifically toll-like receptor-2 (TLR2) and TLR4, using isolated rat alveolar macrophages (aMØs). We assessed the ability of aMØs after brief exposure to acidified saline to elaborate proinflammatory cytokines in response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) stimulation, known ligands of TLR4 and TLR2, respectively. Low pH stress reduced LPS- and LTA-mediated cytokine release (CINC-1, MIP-2, TNF-α, MCP-1, and IFN-ß). LPS and LTA increased intracellular Ca²âº concentrations while Ca²âº chelation by BAPTA decreased LPS- and LTA-mediated cytokine responses. BAPTA blocked the effects of low pH stress on most of LPS-stimulated cytokines but not of LTA-stimulated responses. In vivo mouse model demonstrates suppressed E. coli and S. pneumoniae clearance following acid aspiration. In conclusion, low pH stress inhibits antibacterial cytokine response of aMØs due to impaired TLR2 (MyD88 pathway) and TLR4 signaling (MyD88 and TRIF pathways). The role of Ca²âº in low pH stress-induced signaling is complex but appears to be distinct between LPS- and LTA-mediated responses.

Gastric Aspiration Models.

The procedures described below are for producing gastric aspiration pneumonitis in mice with alterations for rats and rabbits described parenthetically. We use 4 different injury vehicles delivered intratracheally to investigate the inflammatory responses to gastric aspiration: Normal saline (NS) as the injury vehicle controlNS + HCl, pH = 1.25 (acid)NS + gastric particles, pH ≈ 5.3 (part.)NS + gastric particles + HCl, pH = 1.25 (acid + part.) The volume, pH, and gastric particle concentration all affect the resulting lung injury. In mice, we generally use an injury volume of 3.6 ml/kg (rat: 1.2 ml/kg, rabbit: 2.4 ml/kg), an injury pH (for the acid-containing vehicles) of 1.25, and a gastric particulate concentration (in the particulate-containing vehicles) of 10 mg/ml (rat: 40 mg/ml). In our hands this results in a maximal, non-lethal lung injury with ≤ 10% mortality for the most injurious vehicle (i.e., acid + part.) The maximum tolerable particulate concentration needs to be determined empirically for any new strains to be used, especially in genetically-altered mice, because an altered inflammatory response may have detrimental affects on mortality. We have extensive experience utilizing these procedures in the outbred strain, CD-1, as well as many genetically-altered inbred stains on the C57BL/6 background. Choice of strain should be carefully considered, especially in terms of strain-specific immune bias, to assure proper data interpretation. The size of the mouse should be ≥ 20 g at the time of injury. Smaller mice can be attempted, if necessary, but the surgical manipulation becomes increasingly more difficult and the surgery survival rate decreases substantially. There are no size or strain constraints for rat and rabbit models, but we generally use Long-Evans rats at 250-300 g and New Zealand White rats at ≈ 2 kg at the time of initial injury.

Characterization of RNA damage under oxidative stress in Escherichia coli.

We have examined the level of 8-hydroxyguanosine (8-oxo-G), an oxidized form of guanosine, in RNA in Escherichia coli under normal and oxidative stress conditions. The level of 8-oxo-G in RNA rises rapidly and remains high for hours in response to hydrogen peroxide (H2O2) challenge in a dose-dependent manner. H2O2 induced elevation of 8-oxo-G content is much higher in RNA than that of 8-hydroxydeoxyguanosine (8-oxo-dG) in DNA. Under normal conditions, the 8-oxo-G level is low in RNA isolated from the ribosome and it is nearly three times higher in non-ribosomal RNAs. In contrast, 8-oxo-G generated by a short exposure to H2O2 is almost equally distributed in various RNA species, suggesting that although ribosomal RNAs are normally less oxidized, they are not protected against exogenous H2O2. Interestingly, highly folded RNA is not protected from oxidation because 8-oxo-G generated by H2O2 treatment in vitro increases to approximately the same levels in tRNA and rRNA in both native and denatured forms. Lastly, increased RNA oxidation is closely associated with cell death by oxidative stress. Our data suggests that RNA is a primary target for reactive oxygen species and RNA oxidation is part of the paradox that cells have to deal with under oxidative stress.

Novel one-step mechanism for tRNA 3'-end maturation by the exoribonuclease RNase R of Mycoplasma genitalium.

Mycoplasma genitalium is expected to metabolize RNA using unique pathways because its minimal genome encodes very few ribonucleases. In this work, we report that the only exoribonuclease identified in M. genitalium, RNase R, is able to remove tRNA 3'-trailers and generate mature 3'-ends. Several sequence and structural features of a tRNA precursor determine its precise processing at the 3'-end by RNase R in a purified system. The aminoacyl-acceptor stem plays a major role in stopping RNase R digestion at the mature 3'-end. Disruption of the stem causes partial or complete degradation of the pre-tRNA by RNase R, whereas extension of the stem results in the formation of a product terminating downstream at the new mature 3'-end. In addition, the 3'-terminal CCA sequence and the discriminator residue influence the ability of RNase R to stop at the mature 3'-end. RNase R-mediated generation of the mature 3'-end prefers a sequence of RCCN at the 3' terminus of tRNA. Variations of this sequence may cause RNase R to trim further and remove terminal CA residues from the mature 3'-end. Therefore, M. genitalium RNase R can precisely remove the 3'-trailer of a tRNA precursor by recognizing features in the terminal domains of tRNA, a process requiring multiple RNases in most bacteria.