Single-stranded siRNAs activate RNAi in animals.

The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation. Effective ss-siRNAs were identified by iterative design by determining structure-activity relationships correlating chemically modified single strands and Argonaute 2 (AGO2) activities, potency in cells, nuclease stability, and pharmacokinetics. We find that the passenger strand is not necessary for potent gene silencing. The guide-strand activity requires AGO2, demonstrating action through the RNAi pathway. ss-siRNA action requires a 5' phosphate to achieve activity in vivo, and we developed a metabolically stable 5'-(E)-vinylphosphonate (5'-VP) with conformation and sterioelectronic properties similar to the natural phosphate. Identification of potent ss-siRNAs offers an additional option for RNAi therapeutics and an alternate perspective on RNAi mechanism.

Synergistic Targeting of DNA-PK and KIT Signaling Pathways in KIT Mutant Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most common and aggressive form of acute leukemia, with a 5-year survival rate of just 24%. Over a third of all AML patients harbor activating mutations in kinases, such as the receptor tyrosine kinases FLT3 (receptor-type tyrosine-protein kinase FLT3) and KIT (mast/stem cell growth factor receptor kit). FLT3 and KIT mutations are associated with poor clinical outcomes and lower remission rates in response to standard-of-care chemotherapy. We have recently identified that the core kinase of the non-homologous end joining DNA repair pathway, DNA-PK (DNA-dependent protein kinase), is activated downstream of FLT3; and targeting DNA-PK sensitized FLT3-mutant AML cells to standard-of-care therapies. Herein, we investigated DNA-PK as a possible therapeutic vulnerability in KIT mutant AML, using isogenic FDC-P1 mouse myeloid progenitor cell lines transduced with oncogenic mutant KIT (V560G and D816V) or vector control. Targeted quantitative phosphoproteomic profiling identified phosphorylation of DNA-PK in the T2599/T2605/S2608/S2610 cluster in KIT mutant cells, indicative of DNA-PK activation. Accordingly, proliferation assays revealed that KIT mutant FDC-P1 cells were more sensitive to the DNA-PK inhibitors M3814 or NU7441, compared with empty vector controls. DNA-PK inhibition combined with inhibition of KIT signaling using the kinase inhibitors dasatinib or ibrutinib, or the protein phosphatase 2A activators FTY720 or AAL(S), led to synergistic cell death. Global phosphoproteomic analysis of KIT-D816V cells revealed that dasatinib and M3814 single-agent treatments inhibited extracellular signal-regulated kinase and AKT (RAC-alpha serine/threonine-protein kinase)/MTOR (serine/threonine-protein kinase mTOR) activity, with greater inhibition of both pathways when used in combination. Combined dasatinib and M3814 treatment also synergistically inhibited phosphorylation of the transcriptional regulators MYC and MYB. This study provides insight into the oncogenic pathways regulated by DNA-PK beyond its canonical role in DNA repair and demonstrates that DNA-PK is a promising therapeutic target for KIT mutant cancers.

Pharmaco-phosphoproteomic analysis of cancer-associated KIT mutations D816V and V560G.

The CD117 mast/stem cell growth factor receptor tyrosine kinase (KIT) is critical for haematopoiesis, melanogenesis and stem cell maintenance. KIT is commonly activated by mutation in cancers including acute myeloid leukaemia, melanoma and gastrointestinal stromal tumours (GISTs). The kinase and the juxtamembrane domains of KIT are mutation hotspots; with the kinase domain mutation D816V common in leukaemia and the juxtamembrane domain mutation V560G common in GISTs. Given the importance of mutant KIT signalling in cancer, we have conducted a proteomic and phosphoproteomic analysis of myeloid progenitor cells expressing D816V- and V560G-KIT mutants, using an FDCP1 isogenic cell line model. Proteomic analysis revealed increased abundance of proteases and growth signalling proteins in KIT-mutant cells compared to empty vector (EV) controls. Pathway analysis identified increased oxidative phosphorylation in D816V- and V560G-mutant KIT cells, which was targetable using the inhibitor IACS010759. Dysregulation of RNA metabolism and cytoskeleton/adhesion pathways was identified in both the proteome and phosphoproteome of KIT-mutant cells. Phosphoproteome analysis further revealed active kinases such as EGFR, ERK and PKC, which were targetable using pharmacological inhibitors. This study provides a pharmaco-phosphoproteomic profile of D816V- and V560G-mutant KIT cells, which reveals novel therapeutic strategies that may be applicable to a range of cancers.

Proteomic and phosphoproteomic characterisation of primary mouse embryonic fibroblasts.

Fibroblasts are the most common cell type in stroma and function in the support and repair of most tissues. Mouse embryonic fibroblasts (MEFs) are amenable to isolation and rapid growth in culture. MEFs are therefore widely used as a standard model for functional characterisation of gene knockouts, and can also be used in co-cultures, commonly to support embryonic stem cell cultures. To facilitate their use as a research tool, we have performed a comprehensive proteomic and phosphoproteomic characterisation of wild-type primary MEFs from C57BL/6 mice. EIF2/4 and MTOR signalling pathways were abundant in both the proteome and phosphoproteome, along with extracellular matrix (ECM) and cytoskeleton associated pathways. Consistent with this, kinase enrichment analysis identified activation of P38A, P90RSK, P70S6K, and MTOR. Cell surface markers and matrisome proteins were also annotated. Data are available via ProteomeXchange with identifier PXD043244. This provides a comprehensive catalogue of the wild-type MEF proteome and phosphoproteome which can be utilised by the field to guide future work.

Immortalized mouse caput epididymal epithelial (mECap18) cell line recapitulates the in-vivo environment.

Residing between the testes and the vas deferens, the epididymis is a highly convoluted tubule whose unique luminal microenvironment is crucial for the functional maturation of spermatozoa. This microenvironment is created by the combined secretory and resorptive activity of the lining epididymal epithelium, including the release of extracellular vesicles (epididymosomes), which encapsulate fertility modulating proteins and a myriad of small non-coding RNAs (sncRNAs) that are destined for delivery to recipient sperm cells. To enable investigation of this intercellular communication nexus, we have previously developed an immortalized mouse caput epididymal epithelial cell line (mECap18). Here, we describe the application of label-free mass spectrometry to characterize the mECap18 cell proteome and compare this to the proteome of native mouse caput epididymal epithelial cells. We report the identification of 5,313 mECap18 proteins, as many as 75.8% of which were also identified in caput epithelial cells wherein they mapped to broadly similar protein classification groupings. Furthermore, key pathways associated with protein synthesis (e.g., EIF2 signaling) and cellular protection in the male reproductive tract (e.g., sirtuin signaling) were enriched in both proteomes. This comparison supports the utility of the mECap18 cell line as a tractable in-vitro model for studying caput epididymal epithelial cell function.

Prospective Validation of Computed Tomography to Identify Patients at High Risk for Stroke After Transient Ischemic Attack or Minor Stroke.

BACKGROUND: Computed tomography (CT) findings of acute and chronic ischemia are associated with subsequent stroke risk in patients with transient ischemic attack. We sought to validate these associations in a large prospective cohort of patients with transient ischemic attack or minor stroke. METHODS: This prospective cohort study enrolled emergency department patients from 13 hospitals with transient ischemic attack who had CT imaging. Primary outcome was stroke within 90 days. Secondary outcomes were stroke within 2 or 7 days. CT findings were abstracted from radiology reports and classified for the presence of acute ischemia, chronic ischemia, or microangiopathy. Multivariable logistic regression was used to test associations with primary and secondary end points. RESULTS: From 8670 prospectively enrolled patients between May 2010 and May 2017, 8382 had a CT within 24 hours. From this total population, 4547 (54%) patients had evidence of acute ischemia, chronic ischemia, or microangiopathy on CT, of whom 175 had a subsequent stroke within 90 days (3.8% subsequent stroke rate; adjusted odds ratio [aOR], 2.33 [95% CI, 1.62-3.36]). This was in comparison to those with CT imaging without ischemia. Findings associated with an increased risk of stroke at 90 days were isolated acute ischemia (6.0%; aOR, 2.42 [95% CI, 1.03-5.66]), acute ischemia with microangiopathy (10.7%; aOR, 3.34 [95% CI, 1.57-7.14]), chronic ischemia with microangiopathy (5.2%; aOR, 1.83 [95% CI, 1.34-2.50]), and acute ischemia with chronic ischemia and microangiopathy (10.9%; aOR, 3.49 [95% CI, 1.54-7.91]). Acute ischemia with chronic ischemia and microangiopathy were most strongly associated with subsequent stroke within 2 days (aOR, 4.36 [95% CI, 1.31-14.54]) and 7 days (aOR, 4.50 [95% CI, 1.73-11.69]). CONCLUSIONS: In patients with transient ischemic attack or minor stroke, CT evidence of acute ischemia with chronic ischemia or microangiopathy significantly increases the risk of subsequent stroke within 90 days of index visit. The combination of all 3 findings results in the greatest early risk.

Elucidation of the protein composition of mouse seminal vesicle fluid.

The seminal vesicles are male accessory sex glands that contribute the major portion of the seminal plasma in which mammalian spermatozoa are bathed during ejaculation. In addition to conveying sperm through the ejaculatory duct, seminal vesicle secretions support sperm survival after ejaculation, and influence the female reproductive tract to promote receptivity to pregnancy. Analysis of seminal vesicle fluid (SVF) composition by proteomics has proven challenging, due to its highly biased protein signature with a small subset of dominant proteins and the difficulty of solubilizing this viscous fluid. As such, publicly available proteomic datasets identify only 85 SVF proteins in total. To address this limitation, we report a new preparative methodology involving sequential solubilization of mouse SVF in guanidine hydrochloride, acetone precipitation, and analysis by label-free mass spectrometry. Using this strategy, we identified 126 SVF proteins, including 83 previously undetected in SVF. Members of the seminal vesicle secretory protein family were the most abundant, accounting for 79% of all peptide spectrum matches. Functional analysis identified inflammation and formation of the vaginal plug as the two most prominent biological processes. Other notable processes included modulation of sperm function and regulation of the female reproductive tract immune environment. Together, these findings provide a robust methodological framework for future SVF studies and identify novel proteins with potential to influence both male and female reproductive physiology.

Intravenous Iron in Patients Undergoing Maintenance Hemodialysis.

BACKGROUND: Intravenous iron is a standard treatment for patients undergoing hemodialysis, but comparative data regarding clinically effective regimens are limited. METHODS: In a multicenter, open-label trial with blinded end-point evaluation, we randomly assigned adults undergoing maintenance hemodialysis to receive either high-dose iron sucrose, administered intravenously in a proactive fashion (400 mg monthly, unless the ferritin concentration was >700 µg per liter or the transferrin saturation was ≥40%), or low-dose iron sucrose, administered intravenously in a reactive fashion (0 to 400 mg monthly, with a ferritin concentration of <200 µg per liter or a transferrin saturation of <20% being a trigger for iron administration). The primary end point was the composite of nonfatal myocardial infarction, nonfatal stroke, hospitalization for heart failure, or death, assessed in a time-to-first-event analysis. These end points were also analyzed as recurrent events. Other secondary end points included death, infection rate, and dose of an erythropoiesis-stimulating agent. Noninferiority of the high-dose group to the low-dose group would be established if the upper boundary of the 95% confidence interval for the hazard ratio for the primary end point did not cross 1.25. RESULTS: A total of 2141 patients underwent randomization (1093 patients to the high-dose group and 1048 to the low-dose group). The median follow-up was 2.1 years. Patients in the high-dose group received a median monthly iron dose of 264 mg (interquartile range [25th to 75th percentile], 200 to 336), as compared with 145 mg (interquartile range, 100 to 190) in the low-dose group. The median monthly dose of an erythropoiesis-stimulating agent was 29,757 IU in the high-dose group and 38,805 IU in the low-dose group (median difference, -7539 IU; 95% confidence interval [CI], -9485 to -5582). A total of 320 patients (29.3%) in the high-dose group had a primary end-point event, as compared with 338 (32.3%) in the low-dose group (hazard ratio, 0.85; 95% CI, 0.73 to 1.00; P<0.001 for noninferiority; P=0.04 for superiority). In an analysis that used a recurrent-events approach, there were 429 events in the high-dose group and 507 in the low-dose group (rate ratio, 0.77; 95% CI, 0.66 to 0.92). The infection rate was the same in the two groups. CONCLUSIONS: Among patients undergoing hemodialysis, a high-dose intravenous iron regimen administered proactively was superior to a low-dose regimen administered reactively and resulted in lower doses of erythropoiesis-stimulating agent being administered. (Funded by Kidney Research UK; PIVOTAL EudraCT number, 2013-002267-25 .).

Phospho-heavy-labeled-spiketide FAIMS stepped-CV DDA (pHASED) provides real-time phosphoproteomics data to aid in cancer drug selection.

Global high-throughput phosphoproteomic profiling is increasingly being applied to cancer specimens to identify the oncogenic signaling cascades responsible for promoting disease initiation and disease progression; pathways that are often invisible to genomics analysis. Hence, phosphoproteomic profiling has enormous potential to inform and improve individualized anti-cancer treatment strategies. However, to achieve the adequate phosphoproteomic depth and coverage necessary to identify the activated, and hence, targetable kinases responsible for driving oncogenic signaling pathways, affinity phosphopeptide enrichment techniques are required and often coupled with offline high-pressure liquid chromatographic (HPLC) separation prior to nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS). These complex and time-consuming procedures, limit the utility of phosphoproteomics for the analysis of individual cancer patient specimens in real-time, and restrict phosphoproteomics to specialized laboratories often outside of the clinical setting. To address these limitations, here we have optimized a new protocol, phospho-heavy-labeled-spiketide FAIMS Stepped-CV DDA (pHASED), that employs online phosphoproteome deconvolution using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and internal phosphopeptide standards to provide accurate label-free quantitation (LFQ) data in real-time. Compared with traditional single-shot LFQ phosphoproteomics workflows, pHASED provided increased phosphoproteomic depth and coverage (phosphopeptides = 4617 pHASED, 2789 LFQ), whilst eliminating the variability associated with offline prefractionation. pHASED was optimized using tyrosine kinase inhibitor (sorafenib) resistant isogenic FLT3-mutant acute myeloid leukemia (AML) cell line models. Bioinformatic analysis identified differential activation of the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) pathway, responsible for sensing and repairing DNA damage in sorafenib-resistant AML cell line models, thereby uncovering a potential therapeutic opportunity. Herein, we have optimized a rapid, reproducible, and flexible protocol for the characterization of complex cancer phosphoproteomes in real-time, a step towards the implementation of phosphoproteomics in the clinic to aid in the selection of anti-cancer therapies for patients.

In suspected ACS, an unmasked 0/1-h vs. masked 0/3-h hs-cTnT protocol did not differ for death or MI at 1 y.

SOURCE CITATION: Lambrakis K, Papendick C, French JK, et al. Late outcomes of the RAPID-TnT randomized controlled trial: 0/1-hour high-sensitivity troponin T protocol in suspected ACS. Circulation. 2021;144:113-25. 33998255.

Diagnosing the "master mechanism of the universe" in interwar and war-era America.

Drawing on personal testimonials and questions addressed to psychiatric hospital officials, this article explores how patients and their loved ones engaged with the idea of diagnosis in interwar and war-era America. I argue that diagnosis had synergies with intellectual sensibilities of American modernity, among them an enthusiasm for science and newness, a modernist sense of time that could be both forward- and backward-looking, and a knowable, interpreted self. While self-understanding and the creation of life narratives were more often considered the bailiwick of psychoanalysis in this period, understanding subjectivity and self-interpretation were not solely expressed in its conceptual vocabulary. Patient and family dialogs with diagnosis and psychiatric authorities allow for an illumination of the interaction between domestic intuitions, common sense, and folk wisdom, on the one hand, and institutional taxonomy, categorization, and scientific terminology on the other, or more broadly, between dispositions that are ostensibly antimodern and more modern ideas. I suggest that the protean and wide-ranging intellectual origins of the discipline of psychiatry, along with the inherent ambiguity of psychiatric diagnosis during the early 20th century, allowed patients to participate in their own medicalization in the most capacious way possible: by combining biology with diagnostic narrative capacities, as well as broader perceptions of morality and character. In the concluding reflection, I speculate about why it is that late 20th-century American critics and activists have tended to view diagnosis and medicalization as coercive and threatening, in contrast to earlier 20th-century patients and their intimate observers.

Time-resolved proteomic profiling of cigarette smoke-induced experimental chronic obstructive pulmonary disease.

BACKGROUND AND OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is the third leading cause of illness and death worldwide. Current treatments aim to control symptoms with none able to reverse disease or stop its progression. We explored the major molecular changes in COPD pathogenesis. METHODS: We employed quantitative label-based proteomics to map the changes in the lung tissue proteome of cigarette smoke-induced experimental COPD that is induced over 8 weeks and progresses over 12 weeks. RESULTS: Quantification of 7324 proteins enabled the tracking of changes to the proteome. Alterations in protein expression profiles occurred in the induction phase, with 18 and 16 protein changes at 4- and 6-week time points, compared to age-matched controls, respectively. Strikingly, 269 proteins had altered expression after 8 weeks when the hallmark pathological features of human COPD emerge, but this dropped to 27 changes at 12 weeks with disease progression. Differentially expressed proteins were validated using other mouse and human COPD bronchial biopsy samples. Major changes in RNA biosynthesis (heterogeneous nuclear ribonucleoproteins C1/C2 [HNRNPC] and RNA-binding protein Musashi homologue 2 [MSI2]) and modulators of inflammatory responses (S100A1) were notable. Mitochondrial dysfunction and changes in oxidative stress proteins also occurred. CONCLUSION: We provide a detailed proteomic profile, identifying proteins associated with the pathogenesis and disease progression of COPD establishing a platform to develop effective new treatment strategies.

Accelerated diagnostic algorithms with hs-cTn have ≥ 92% sensitivity but lower specificity for diagnosing AMI.

SOURCE CITATION: Lee CC, Huang SS, Yeo YH, et al. High-sensitivity-cardiac troponin for accelerated diagnosis of acute myocardial infarction: a systematic review and meta-analysis. Am J Emerg Med. 2019. [Epub ahead of print]. 31932131.

Healing Rates of Venous Leg Ulcers Managed With Compression Therapy: A Secondary Analysis of Data.

PURPOSE: The purpose of this study was to explore average time to heal for patients with venous leg ulcers (VLUs) receiving standard of care that included compression and advanced wound dressings. DESIGN: Secondary analysis of an existing electronic database. SUBJECT AND SETTINGS: A convenience sample consisting of 1323 patients with VLUs from various community care sectors (homecare and clinics) across Canada. METHODS: The Wound Studies database used in the analysis consisted of data from 6 studies conducted prospectively between 1999 and 2009 in which the treatment and delivery of care for all lower leg ulcers (venous, arterial, and mixed) in Canada was examined. From these studies, only patients with VLUs, with an ankle-brachial pressure index of greater than 0.8, and surface area measurements of the ulcers at baseline, 3 months, and 6 months were included. Descriptive statistics were used to determine the proportion of patients who achieved closure at 3 and 6 months and explore the weekly and monthly healing rates for those who did and did not achieve closure. Logistic regression analysis was performed to identify predictive factors for healing. RESULTS: A total of 777 patients (mean age 69 years) met inclusion criteria. The proportion of patients who achieved closure at 3 and 6 months was 42.2% and 48.6%, respectively. Of the participants who achieved wound closure, monthly mean healing rate, measured by percentage of reduction in surface area, was 33.4% (0.56 cm, SD 1.4 [median 0.15 cm]) through month 3, and 31.0% (0.70 cm, SD 1.6 [median 0.08 cm]) through month 6. The overall monthly surface area reduction was 30%. CONCLUSION: Study findings suggest a monthly surface area reduction of 30% provides a baseline healing rate for VLUs managed with compression therapy and advanced dressings. Findings also suggest standard of care is not sufficient for healing in over 50% of the population, as the proportion of those who achieved closure at 3 and 6 months was 42.2% and 48.6%, respectively.

Web-based physiotherapy for people affected by multiple sclerosis: a single blind, randomized controlled feasibility study.

OBJECTIVE:: To examine the feasibility of a trial to evaluate web-based physiotherapy compared to a standard home exercise programme in people with multiple sclerosis. DESIGN:: Multi-centre, randomized controlled, feasibility study. SETTING:: Three multiple sclerosis out-patient centres. PARTICIPANTS:: A total of 90 people with multiple sclerosis (Expanded Disability Status Scale 4-6.5). INTERVENTIONS:: Participants were randomized to a six-month individualized, home exercise programme delivered via web-based physiotherapy ( n = 45; intervention) or a sheet of exercises ( n = 45; active comparator). OUTCOME MEASURES:: Outcome measures (0, three, six and nine months) included adherence, two-minute walk test, 25 foot walk, Berg Balance Scale, physical activity and healthcare resource use. Interviews were undertaken with 24 participants and 3 physiotherapists. RESULTS:: Almost 25% of people approached agreed to take part. No intervention-related adverse events were recorded. Adherence was 40%-63% and 53%-71% in the intervention and comparator groups. There was no difference in the two-minute walk test between groups at baseline (Intervention-80.4(33.91)m, Comparator-70.6(31.20)m) and no change over time (at six-month Intervention-81.6(32.75)m, Comparator-74.8(36.16)m. There were no significant changes over time in other outcome measures except the EuroQol-5 Dimension at six months which decreased in the active comparator group. For a difference of 8(17.4)m in two-minute walk test between groups, 76 participants/group would be required (80% power, P > 0.05) for a future randomized controlled trial. CONCLUSION:: No changes were found in the majority of outcome measures over time. This study was acceptable and feasible by participants and physiotherapists. An adequately powered study needs 160 participants.

Randomized Trial Comparing Proactive, High-Dose versus Reactive, Low-Dose Intravenous Iron Supplementation in Hemodialysis (PIVOTAL): Study Design and Baseline Data.

BACKGROUND: Intravenous (IV) iron supplementation is a standard maintenance treatment for hemodialysis (HD) patients, but the optimum dosing regimen is unknown. METHODS: PIVOTAL (Proactive IV irOn Therapy in hemodiALysis patients) is a multicenter, open-label, blinded endpoint, randomized controlled (PROBE) trial. Incident HD adults with a serum ferritin < 400 µg/L and transferrin saturation (TSAT) levels < 30% receiving erythropoiesis-stimulating agents (ESA) were eligible. Enrolled patients were randomized to a proactive, high-dose IV iron arm (iron sucrose 400 mg/month unless ferritin > 700 µg/L and/or TSAT ≥40%) or a reactive, low-dose IV iron arm (iron sucrose administered if ferritin <200 µg/L or TSAT < 20%). We hypothesized that proactive, high-dose IV iron would be noninferior to reactive, low-dose IV iron for the primary outcome of first occurrence of nonfatal myocardial infarction (MI), nonfatal stroke, hospitalization for heart failure or death from any cause. If noninferiority is confirmed with a noninferiority limit of 1.25 for the hazard ratio of the proactive strategy relative to the reactive strategy, a test for superiority will be carried out. Secondary outcomes include infection-related endpoints, ESA dose requirements, and quality-of-life measures. As an event-driven trial, the study will continue until at least 631 primary outcome events have accrued, but the expected duration of follow-up is 2-4 years. RESULTS: Of the 2,589 patients screened across 50 UK sites, 2,141 (83%) were randomized. At baseline, 65.3% were male, the median age was 65 years, and 79% were white. According to eligibility criteria, all patients were on ESA at screening. Prior stroke and MI were present in 8 and 9% of the cohort, respectively, and 44% of patients had diabetes at baseline. Baseline data for the randomized cohort were generally concordant with recent data from the UK Renal Registry. CONCLUSIONS: PIVOTAL will provide important information about the optimum dosing of IV iron in HD patients representative of usual clinical practice. TRIAL REGISTRATION: EudraCT number: 2013-002267-25.

Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function.

Viable constitutive and tamoxifen inducible liver-specific RNase H1 knockout mice that expressed no RNase H1 activity in hepatocytes showed increased R-loop levels and reduced mitochondrial encoded DNA and mRNA levels, suggesting impaired mitochondrial R-loop processing, transcription and mitochondrial DNA replication. These changes resulted in mitochondrial dysfunction with marked changes in mitochondrial fusion, fission, morphology and transcriptional changes reflective of mitochondrial damage and stress. Liver degeneration ensued, as indicated by apoptosis, fibrosis and increased transaminase levels. Antisense oligonucleotides (ASOs) designed to serve as substrates for RNase H1 were inactive in the hepatocytes from the RNase H1 knockout mice and in vivo, demonstrating that RNase H1 is necessary for the activity of DNA-like ASOs. During liver regeneration, a clone of hepatocytes that expressed RNase H1 developed and partially restored mitochondrial and liver function.