Surgical treatment of refractory low back pain using implanted BurstDR spinal cord stimulation (SCS) in a cohort of patients without options for corrective surgery: Findings and results from the DISTINCT study, a prospective randomized multi-center-controlled trial.

Background: Low back pain (LBP) is a highly prevalent, disabling condition affecting millions of people. Patients with an identifiable anatomic pain generator and resulting neuropathic lower extremity symptoms often undergo spine surgery, but many patients lack identifiable and/or surgically corrective pathology. Nonoperative treatment options often fail to provide sustained relief. Spinal cord stimulation (SCS) is sometimes used to treat these patients, but the lack of level 1 evidence limits its widespread use and insurance coverage. The DISTINCT RCT study evaluates the efficacy of passive recharge burst SCS compared to conventional medical treatment (CMM) in alleviating chronic, refractory axial low back pain. Methods: This prospective, multicenter, randomized, study with an optional 6-month crossover involved patients who were not candidates for lumbar spine surgery. The primary and secondary endpoints evaluated improvements in low back pain intensity (NRS), back pain-related disability (ODI), pain catastrophizing (PCS), and healthcare utilization. Patients were randomized to SCS therapy or CMM at 30 US study sites. Results: The SCS arm reported an 85.3% NRS responder rate (≥ 50% reduction) compared to 6.2% (5/81) in the CMM arm. After the 6M primary endpoint, SCS patients elected to remain on assigned therapy and 66.2% (49/74) of CMM patients chose to trial SCS (crossover). At the 12M follow-up, SCS and crossover patients reported 78.6% and 71.4% NRS responder rates. Secondary outcomes indicated significant improvements in ODI, PCS, and reduced healthcare utilization. Six serious adverse events were reported and resolved without sequelae. Conclusion: DISTINCT chronic low back pain patients with no indication for corrective surgery experienced a significant and sustained response to burst SCS therapy for up to 12 months. CMM patients who crossed over to the SCS arm reported profound improvements after 6 months. This data advocates for a timely consideration of SCS therapy in patients unresponsive to conservative therapy.

Randomized Placebo-Controlled Trial of 60-Day Percutaneous Peripheral Nerve Stimulation Treatment Indicates Relief of Persistent Postoperative Pain, and Improved Function After Knee Replacement.

OBJECTIVES: Total knee arthroplasty (TKA) is an effective surgery for end-stage knee osteoarthritis, but chronic postoperative pain and reduced function affect up to 20% of patients who undergo such surgery. There are limited treatment options, but percutaneous peripheral nerve stimulation (PNS) is a promising nonopioid treatment option for chronic, persistent postoperative pain. The objective of the present study was to evaluate the effect of a 60-day percutaneous PNS treatment in a multicenter, randomized, double-blind, placebo-controlled trial for treating persistent postoperative pain after TKA. MATERIALS AND METHODS: Patients with postoperative pain after knee replacement were screened for this postmarket, institutional review board-approved, prospectively registered (NCT04341948) trial. Subjects were randomized to receive either active PNS or placebo (sham) stimulation. Subjects and a designated evaluator were blinded to group assignments. Subjects in both groups underwent ultrasound-guided placement of percutaneous fine-wire coiled leads targeting the femoral and sciatic nerves on the leg with postoperative pain. Leads were indwelling for eight weeks, and the primary efficacy outcome compared the proportion of subjects in each group reporting ≥50% reduction in average pain relative to baseline during weeks five to eight. Functional outcomes (6-minute walk test; 6MWT and Western Ontario and McMaster Universities Osteoarthritis Index) and quality of life (Patient Global Impression of Change) also were evaluated at end of treatment (EOT). RESULTS: A greater proportion of subjects in the PNS groups (60%; 12/20) than in the placebo (sham) group (24%; 5/21) responded with ≥50% pain relief relative to baseline (p = 0.028) during the primary endpoint (weeks 5-8). Subjects in the PNS group also walked a significantly greater distance at EOT than did those in the placebo (sham) group (6MWT; +47% vs -9% change from baseline; p = 0.048, n = 18 vs n = 20 completed the test, respectively). Prospective follow-up to 12 months is ongoing. CONCLUSIONS: This study provides evidence that percutaneous PNS decreases persistent pain, which leads to improved functional outcomes after TKA at EOT.

Treatment of Refractory Low Back Pain Using Passive Recharge Burst in Patients Without Options for Corrective Surgery: Findings and Results From the DISTINCT Study, a Prospective Randomized Multicenter Controlled Trial.

OBJECTIVE: Spinal cord stimulation (SCS) is effective for relieving chronic intractable pain conditions. The Dorsal spInal cord STImulatioN vs mediCal management for the Treatment of low back pain study evaluates the effectiveness of SCS compared with conventional medical management (CMM) in the treatment of chronic low back pain in patients who had not undergone and were not candidates for lumbar spine surgery. METHODS AND MATERIALS: Patients were randomized to passive recharge burst therapy (n = 162) or CMM (n = 107). They reported severe pain and disability for more than a decade and had failed a multitude of therapies. Common diagnoses included degenerative disc disease, spondylosis, stenosis, and scoliosis-yet not to a degree amenable to surgery. The six-month primary end point compared responder rates, defined by a 50% reduction in pain. Hierarchical analyses of seven secondary end points were performed in the following order: composite responder rate (numerical rating scale [NRS] or Oswestry Disability Index [ODI]), NRS, ODI, Pain Catastrophizing Scale responder rate, Patient Global Impression of Change (PGIC) responder rate, and Patient-Reported Outcome Measure Information System-29 in pain interference and physical function. RESULTS: Intention-to-treat analysis showed a significant difference in pain responders on NRS between SCS (72.6%) and CMM (7.1%) arms (p < 0.0001). Of note, 85.2% of those who received six months of therapy responded on NRS compared with 6.2% of those with CMM (p < 0.0001). All secondary end points indicated the superiority of burst therapy over CMM. A composite measure on function or pain relief showed 91% of subjects with SCS improved, compared with 16% of subjects with CMM. A substantial improvement of 30 points was observed on ODI compared with a

Primary Ewing's Sarcoma of the Penis: First Reported Case in the United Kingdom.

Penile cancer is generally rare, and Squamous cell cancer of the penis is the most common histological type. Sarcoma of the penis has a low incidence, but they tend to grow faster than other penile cancers. One of the rarest types of penile sarcomas is Extra-Skeletal Ewing's Sarcoma (EES). The management of such cases can be challenging, and treatment guidelines do not exist for these rare cases. We present a rare case of EES that has developed in the penis of a young patient in the United Kingdom.

Male urethral stricture disease: why management guidelines are challenging in low-income countries.

Urethral stricture disease (USD) is one of the commonest urological pathologies in adult men in low- or low-middle-income countries, providing a significant work burden for the small number of specialist surgeons who are able to provide appropriate treatment. The underlying causes of anterior urethral stricture relate to urethral fibrosis from sexually transmitted infection, with posterior urethral disruption secondary to pelvic trauma being an equally common cause of USD in many countries in sub-Saharan Africa. Anterior urethral strictures are often long, and multifocal, and bulbo-prostatic disruptions are usually due to relatively low-velocity pelvic trauma. The management options available in resource-poor settings are often severely limited by the individual's ability to pay for care, the availability of a specialist surgeon and, importantly, a shortage of functioning endoscopic equipment for less-invasive treatments. Consequently, reconstructive surgery is often regarded by the patient, and surgeon, as the most cost-effective and, therefore, primary means of treating a urethral stricture once urethral dilatation has failed. Regional anaesthetic techniques have limited the adoption of free-graft augmentation as an alternative to pedicled flaps of locally available skin for reconstruction, whilst an inability to provide tension-free bulbo-prostatic anastomoses has negatively impacted the outcome from the treatment of pelvic fracture disruption injuries in much of sub-Saharan Africa. However, Urolink has found that local surgeons can be taught sustainable skills required for successful complex urethroplasty when supported by longitudinal mentorship in the management of difficult clinical issues. Evidence-based practice is known to improve the standard of care in specific conditions in high-income countries, including the management of male USD. However, guidelines developed in high-income countries are not necessarily appropriate for stricture management in less well-resourced healthcare environments but could be adapted to help improve the delivery of stricture care for men in low- or low-middle income countries.

Scrotal Masson's tumour masquerading as dysplastic polyorchidism - a case report.

This case describes a rare genitourinary presentation of a Masson's tumour, preoperatively assumed to be a dysplastic third testis.

Clinical Practice Guideline: Opioid Prescribing for Analgesia After Common Otolaryngology Operations.

OBJECTIVE: Opioid use disorder (OUD), which includes the morbidity of dependence and mortality of overdose, has reached epidemic proportions in the United States. Overprescription of opioids can lead to chronic use and misuse, and unused narcotics after surgery can lead to their diversion. Research supports that most patients do not take all the prescribed opioids after surgery and that surgeons are the second largest prescribers of opioids in the United States. The introduction of opioids in those with OUD often begins with prescription opioids. Reducing the number of extra opioids available after surgery through smaller prescriptions, safe storage, and disposal should reduce the risk of opioid use disorder in otolaryngology patients and their families. PURPOSE: The purpose of this specialty-specific guideline is to identify quality improvement opportunities in postoperative pain management of common otolaryngologic surgical procedures. These opportunities are communicated through clear actionable statements with explanation of the support in the literature, evaluation of the quality of the evidence, and recommendations on implementation. Employing these action statements should reduce the variation in care across the specialty and improve postoperative pain control while reducing risk of OUD. The target patients for the guideline are any patients treated for anticipated or reported pain within the first 30 days after undergoing common otolaryngologic procedures. The target audience of the guideline is otolaryngologists who perform surgery and clinicians who manage pain after surgical procedures. Outcomes to be considered include whether the patient has stopped using opioids, has disposed of unused opioids, and was satisfied with the pain management plan.The guideline addresses assessment of the patient for OUD risk factors, counseling on pain expectations, and identifying factors that can affect pain duration and/or severity. It also discusses the use of multimodal analgesia as first-line treatment and the responsible use of opioids. Last, safe disposal of unused opioids is discussed.This guideline is intended to focus on evidence-based quality improvement opportunities judged most important by the guideline development group. It is not a comprehensive guide on pain management in otolaryngologic procedures. The statements in this guideline are not intended to limit or restrict care provided by clinicians based on their experiences and assessments of individual patients. ACTION STATEMENTS: The guideline development group made strong recommendations for the following key action statements: (3A) prior to surgery, clinicians should identify risk factors for opioid use disorder when analgesia using opioids is anticipated; (6) clinicians should advocate for nonopioid medications as first-line management of pain after otolaryngologic surgery; (9) clinicians should recommend that patients (or their caregivers) store prescribed opioids securely and dispose of unused opioids through take-back programs or another accepted method.The guideline development group made recommendations for the following key action statements: (1) prior to surgery, clinicians should advise patients and others involved in the postoperative care about the expected duration and severity of pain; (2) prior to surgery, clinicians should gather information specific to the patient that modifies severity and/or duration of pain; (3B) in patients at risk for OUD, clinicians should evaluate the need to modify the analgesia plan; (4) clinicians should promote shared decision making by informing patients of the benefits and risks of postoperative pain treatments that include nonopioid analgesics, opioid analgesics, and nonpharmacologic interventions; (5) clinicians should develop a multimodal treatment plan for managing postoperative pain; (7) when treating postoperative pain with opioids, clinicians should limit therapy to the lowest effective dose and the shortest duration; (8A) clinicians should instruct patients and caregivers how to communicate if pain is not controlled or if medication side effects occur; (8B) clinicians should educate patients to stop opioids when pain is controlled with nonopioids and stop all analgesics when pain has resolved; (10) clinicians should inquire, within 30 days of surgery, whether the patient has stopped using opioids, has disposed of unused opioids, and was satisfied with the pain management plan.

Clinical Practice Guideline: Opioid Prescribing for Analgesia After Common Otolaryngology Operations Executive Summary.

OBJECTIVE: Opioid use disorder (OUD), which includes the morbidity of dependence and mortality of overdose, has reached epidemic proportions in the United States. Overprescription of opioids can lead to chronic use and misuse, and unused narcotics after surgery can lead to their diversion. Research supports that most patients do not take all the prescribed opioids after surgery and that surgeons are the second largest prescribers of opioids in the United States. The introduction of opioids in those with OUD often begins with prescription opioids. Reducing the number of extra opioids available after surgery through smaller prescriptions, safe storage, and disposal should reduce the risk of opioid use disorder in otolaryngology patients and their families. PURPOSE: The purpose of this specialty-specific guideline is to identify quality improvement opportunities in postoperative pain management of common otolaryngologic surgical procedures. These opportunities are communicated through clear actionable statements with explanation of the support in the literature, evaluation of the quality of the evidence, and recommendations on implementation. Employing these action statements should reduce the variation in care across the specialty and improve postoperative pain control while reducing risk of OUD. The target patients for the guideline are any patients treated for anticipated or reported pain within the first 30 days after undergoing common otolaryngologic procedures. The target audience of the guideline is otolaryngologists who perform surgery and clinicians who manage pain after surgical procedures. Outcomes to be considered include whether the patient has stopped using opioids, has disposed of unused opioids, and was satisfied with the pain management plan.The guideline addresses assessment of the patient for OUD risk factors, counseling on pain expectations, and identifying factors that can affect pain duration and/or severity. It also discusses the use of multimodal analgesia as first-line treatment and the responsible use of opioids. Last, safe disposal of unused opioids is discussed.This guideline is intended to focus on evidence-based quality improvement opportunities judged most important by the guideline development group. It is not a comprehensive guide on pain management in otolaryngologic procedures. The statements in this guideline are not intended to limit or restrict care provided by clinicians based on their experiences and assessments of individual patients. ACTION STATEMENTS: The guideline development group made strong recommendations for the following key action statements: (3A) prior to surgery, clinicians should identify risk factors for opioid use disorder when analgesia using opioids is anticipated; (6) clinicians should advocate for nonopioid medications as first-line management of pain after otolaryngologic surgery; (9) clinicians should recommend that patients (or their caregivers) store prescribed opioids securely and dispose of unused opioids through take-back programs or another accepted method.The guideline development group made recommendations for the following key action statements: (1) prior to surgery, clinicians should advise patients and others involved in the postoperative care about the expected duration and severity of pain; (2) prior to surgery, clinicians should gather information specific to the patient that modifies severity and/or duration of pain; (3B) in patients at risk for OUD, clinicians should evaluate the need to modify the analgesia plan; (4) clinicians should promote shared decision making by informing patients of the benefits and risks of postoperative pain treatments that include nonopioid analgesics, opioid analgesics, and nonpharmacologic interventions; (5) clinicians should develop a multimodal treatment plan for managing postoperative pain; (7) when treating postoperative pain with opioids, clinicians should limit therapy to the lowest effective dose and the shortest duration; (8A) clinicians should instruct patients and caregivers how to communicate if pain is not controlled or if medication side effects occur; (8B) clinicians should educate patients to stop opioids when pain is controlled with nonopioids and stop all analgesics when pain has resolved; (10) clinicians should inquire, within 30 days of surgery, whether the patient has stopped using opioids, has disposed of unused opioids, and was satisfied with the pain management plan.

Postoperative Pain and Analgesic Requirements in the First Year after Intraoperative Methadone for Complex Spine and Cardiac Surgery.

BACKGROUND: Methadone is a long-acting opioid that has been reported to reduce postoperative pain scores and analgesic requirements and may attenuate development of chronic postsurgical pain. The aim of this secondary analysis of two previous trials was to follow up with patients who had received a single intraoperative dose of either methadone or traditional opioids for complex spine or cardiac surgical procedures. METHODS: Preplanned analyses of long-term outcomes were conducted for spinal surgery patients randomized to receive 0.2 mg/kg methadone at the start of surgery or 2 mg hydromorphone at surgical closure, and for cardiac surgery patients randomized to receive 0.3 mg/kg methadone or 12 µg/kg fentanyl intraoperatively. A pain questionnaire assessing the weekly frequency (the primary outcome) and intensity of pain was mailed to subjects 1, 3, 6, and 12 months after surgery. Ordinal data were compared with the Mann-Whitney U test, and nominal data were compared using the chi-square test or Fisher exact probability test. The criterion for rejection of the null hypothesis was P < 0.01. RESULTS: Three months after surgery, patients randomized to receive methadone for spine procedures reported the weekly frequency of chronic pain was less (median score 0 on a 0 to 4 scale [less than once a week] vs. 3 [daily] in the hydromorphone group, P = 0.004). Patients randomized to receive methadone for cardiac surgery reported the frequency of postsurgical pain was less at 1 month (median score 0) than it was in patients randomized to receive fentanyl (median score 2 [twice per week], P = 0.004). CONCLUSIONS: Analgesic benefits of a single dose of intraoperative methadone were observed during the first 3 months after spinal surgery (but not at 6 and 12 months), and during the first month after cardiac surgery, when the intensity and frequency of pain were the greatest.

Pharmacogenomic considerations for medications in the perioperative setting.

Several high-profile examples of adverse outcomes from medications used in the perioperative setting are well known (e.g., malignant hyperthermia, prolonged apnea, respiratory depression, inadequate analgesia), leading to an increased understanding of genetic susceptibilities underlying these risks. Pharmacogenomic information is increasingly being utilized in certain areas of medicine. Despite this, routine preoperative genetic screening to inform medication risk is not yet standard practice. In this review, we assess the current readiness of pharmacogenomic information for clinical consideration for several common perioperative medications, including description of key pharmacogenes, pharmacokinetic implications and potential clinical outcomes. The goal is to highlight medications for which emerging or considerable pharmacogenomic information exists and identify areas for future potential research.

The impact of psychiatric comorbidities on patient-reported surgical outcomes in adults treated for the median arcuate ligament syndrome.

OBJECTIVE: Median arcuate ligament syndrome (MALS) is an often overlooked, surgically correctable condition that mimics functional chronic abdominal pain. Patient-reported surgical outcomes are unpredictable in MALS. The objective of this study was to define the psychiatric comorbidities in a cohort of adults undergoing surgery for MALS and to determine whether these comorbidities are predictive of patient-reported quality of life (QOL) outcomes. METHODS: A prospective observational trial was conducted between April 1, 2010, and December 31, 2015, at a single tertiary care hospital. Adults with a diagnosis of chronic abdominal pain in the setting of celiac artery compression were enrolled in a prospective Institutional Review Board-approved observational trial. Patients completed psychological assessments before surgery for MALS and at 6 months after surgery. The primary outcome was patient-reported health-related QOL (young adult version of the Pediatric Quality of Life Inventory). RESULTS: A total of 51 patients (80% female; n = 41) with a mean age of 30.5 (±12.4) years were enrolled. Surgery significantly improved celiac artery hemodynamics in the entire cohort (P < .0001) as well as overall QOL (67.8 ± 14.6 [before surgery] vs 80.3 ± 13.7 [after surgery]; P < .001). Psychiatric diagnoses were common in this cohort, with 14 of 51 (28%) patients meeting criteria for a psychiatric diagnosis. There were no differences in the number of patients with psychiatric diagnoses between presurgical and postsurgical evaluations (14 [28%] vs 13 [26%]; P = .8). Exploratory analyses suggest that having a psychiatric diagnosis at the presurgical evaluation may predict significantly lower postsurgical QOL (R2 = 0.009; P = .01). CONCLUSIONS: Surgery improves patient-reported QOL in adults treated for MALS. Psychiatric diagnoses are common in adults with MALS and predict worse patient-reported QOL outcomes.

Local anesthetic systemic toxicity.

Local anesthetic systemic toxicity (LAST) is a rare yet devastating complication from the administration of local anesthesia. The ability to recognize and treat LAST is critical for clinicians who administer these drugs. The authors reviewed the literature on the mechanism, treatment, and prevention of LAST, with the goal of proposing a practical method for its management.

Acute pain management.

This article updates acute pain management in ambulatory surgery and proposes a practical three-step approach for reducing the impact and incidence of uncontrolled surgical pain. By identifying at-risk patients, implementing multimodal analgesia, and intervening promptly with rescue therapies, the anesthesiologist may improve outcomes, reduce cost, and optimize the patient's experience and quality of recovery.

New developments in the treatment of localized penile cancer.

OBJECTIVES: To analyze the current trends in local therapy approaches in patients with penile carcinoma. METHODS: The relevant published data since 2000 were reviewed; important series published before 2000 were also included. The reports were classified according to the level of evidence. Review studies and others indirectly related to the topic were also included but not classified. RESULTS: New information has suggested that surgical margins of only a few millimeters might be adequate for most localized tumors. A trend toward the use of more conservative therapies instead of amputative surgery has been observed, especially in developed countries. Although the local recurrence rate has been greater after conservative therapies than after amputative surgery, this increased rate does not seemed to have had a negative effect on cancer-specific survival. The quality of life has been superior after conservative procedures with preservation of the penis that seems to give the best results with regard to sexual function. Reconstructive surgery can be performed in selected patients after amputative surgery. CONCLUSIONS: Although the level of evidence is low, conservative therapies can be recommended for selected patients with penile carcinoma. Despite the trend for conservative approaches, these patients need psychological support.

Comparison of HIV-derived lentiviral and MLV-based gammaretroviral vector integration sites in primate repopulating cells.

The potential for leukemia caused by retroviral vector integration has become a significant concern for hematopoietic stem cell gene therapy. We analyzed the distribution of vector integrants in pigtailed macaque and baboon repopulating cells for the two most commonly used retroviral vector systems, human immunodeficiency virus (HIV)-based lentiviral vectors and murine leukemia virus (MLV)-based gammaretroviral vectors, to help define their relative genotoxicity. All animals had polyclonal engraftment with no apparent adverse effects from transplantation with gene-modified cells. In all, 380 MLV and 235 HIV unique vector integration sites were analyzed and had distinct distribution patterns in relation to genes and CpG islands as observed in previous in vitro studies. Both vector types were found more frequently in and near proto-oncogenes in repopulating cells than in a random dataset. Analysis of functional classes of genes with integrants within 100 kilobases (kb) of their transcription start sites showed an over-representation of genes involved in growth or survival near both lentiviral and gammaretroviral integrants. Microarray analysis showed that both gammaretroviral and lentiviral vectors were found close to genes with high expression levels in primitive cells enriched for hematopoietic stem cells. These data help define the relative risk of insertional mutagenesis with MLV-, HIV-, and simian immunodeficiency virus (SIV)-based vectors in a highly relevant primate model.

An unmethylated 3' promoter-proximal region is required for efficient transcription initiation.

The promoter regions of approximately 40% of genes in the human genome are embedded in CpG islands, CpG-rich regions that frequently extend on the order of one kb 3' of the transcription start site (TSS) region. CpGs 3' of the TSS of actively transcribed CpG island promoters typically remain methylation-free, indicating that maintaining promoter-proximal CpGs in an unmethylated state may be important for efficient transcription. Here we utilize recombinase-mediated cassette exchange to introduce a Moloney Murine Leukemia Virus (MoMuLV)-based reporter, in vitro methylated 1 kb downstream of the TSS, into a defined genomic site. In a subset of clones, methylation spreads to within approximately 320 bp of the TSS, yielding a dramatic decrease in transcript level, even though the promoter/TSS region remains unmethylated. Chromatin immunoprecipitation analyses reveal that such promoter-proximal methylation results in loss of RNA polymerase II and TATA-box-binding protein (TBP) binding in the promoter region, suggesting that repression occurs at the level of transcription initiation. While DNA methylation-dependent trimethylation of H3 lysine (K)9 is confined to the intragenic methylated region, the promoter and downstream regions are hypo-acetylated on H3K9/K14. Furthermore, DNase I hypersensitivity and methylase-based single promoter analysis (M-SPA) experiments reveal that a nucleosome is positioned over the unmethylated TATA-box in these clones, indicating that dense DNA methylation downstream of the promoter region is sufficient to alter the chromatin structure of an unmethylated promoter. Based on these observations, we propose that a DNA methylation-free region extending several hundred bases downstream of the TSS may be a prerequisite for efficient transcription initiation. This model provides a biochemical explanation for the typical positioning of TSSs well upstream of the 3' end of the CpG islands in which they are embedded.

DNA methylation density influences the stability of an epigenetic imprint and Dnmt3a/b-independent de novo methylation.

DNA methylation plays an important role in transcriptional repression. To gain insight into the dynamics of demethylation and de novo methylation, we introduced a proviral reporter, premethylated at different densities, into a defined chromosomal site in murine erythroleukemia cells and monitored the stability of the introduced methylation and reporter gene expression. A high density of methylation was faithfully propagated in vivo. In contrast, a low level of methylation was not stable, with complete demethylation and associated transcriptional activation or maintenance-coupled de novo methylation and associated silencing occurring with equal probability. Deletion of the proviral enhancer increased the probability of maintenance-coupled de novo methylation, suggesting that this enhancer functions in part to antagonize such methylation. The DNA methyltransferases (MTases) Dnmt3a and Dnmt3b are thought to be the sole de novo MTases in the mammalian genome. To determine whether these enzymes are responsible for maintenance-coupled de novo methylation, the unmethylated or premethylated proviral reporter was introduced into DNA MTase-deficient embryonic stem cells. These studies revealed the presence of a Dnmt3a/Dnmt3b-independent de novo methyltransferase activity that is stimulated by the presence of preexisting methylation.