Basilar Impression: A Systematic Review and Meta-Analysis of Clinical Features, Operative Strategies and Outcomes.

OBJECTIVE: Basilar Impression (BI) is a rare yet debilitating abnormality of the craniovertebral junction (CVJ), known to cause life-threatening medullary brainstem compression. Our study analyzes surgical approaches for BI and related outcomes. METHODS: A systematic review was conducted using PubMed, Google Scholar, and Web of Science electronic databases according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to critically assess primary articles examining BI. RESULTS: We analyzed 87 patients from 65 articles, mostly female (55.17%) with a mean age of 46.31 ± 17.94 years, commonly presenting with motor (59.77%) and sensory deficits (55.17%). Commonly employed procedures included posterior occipitocervical fusion (24.14%), anterior decompression (20.69%), and combined anterior decompression with posterior fusion (21.84%). Patients who underwent anterior approaches were found to be older (55.38 ± 17.67 vs. 45.49 ± 18.78 years, p < 0.05) and had a longer duration from symptom onset to surgery (57.39 ± 64.33 vs. 26.02 ± 29.60 months, p < 0.05) compared to posterior approaches. Our analysis revealed a significant association between a longer duration from symptom onset to surgery and an increased likelihood of undergoing odontoidectomy and decompression (OR: 1.02, 95% CI: 1.00-1.03, p < 0.05). Furthermore, after adjusting for all other covariates, a history of rheumatoid arthritis and the use of a posterior approach were significantly associated with an elevated risk of postoperative complications (p < 0.05). CONCLUSIONS: The treatment approach to complex CVJ disease should be tailored to the surgeon's experience and the nature of the compressive pathology.

Isolated Traumatic Subarachnoid Hemorrhage on Head Computed Tomography Scan May Not Be Isolated: A Transforming Research and Clinical Knowledge in Traumatic Brain Injury Study (TRACK-TBI) Study.

Isolated traumatic subarachnoid hemorrhage (tSAH) after traumatic brain injury (TBI) on head computed tomography (CT) scan is often regarded as a "mild" injury, with reduced need for additional workup. However, tSAH is also a predictor of incomplete recovery and unfavorable outcome. This study aimed to evaluate the characteristics of CT-occult intracranial injuries on brain magnetic resonance imaging (MRI) scan in TBI patients with emergency department (ED) arrival Glasgow Coma Scale (GCS) score 13-15 and isolated tSAH on CT. The prospective, 18-center Transforming Research and Clinical Knowledge in Traumatic Brain Injury Study (TRACK-TBI; enrollment years 2014-2019) enrolled participants who presented to the ED and received a clinically-indicated head CT within 24 h of TBI. A subset of TRACK-TBI participants underwent venipuncture within 24 h for plasma glial fibrillary acidic protein (GFAP) analysis, and research MRI at 2-weeks post-injury. In the current study, TRACK-TBI participants age ≥17 years with ED arrival GCS 13-15, isolated tSAH on initial head CT, plasma GFAP level, and 2-week MRI data were analyzed. In 57 participants, median age was 46.0 years [quartile 1 to 3 (Q1-Q3): 34-57] and 52.6% were male. At ED disposition, 12.3% were discharged home, 61.4% were admitted to hospital ward, and 26.3% to intensive care unit. MRI identified CT-occult traumatic intracranial lesions in 45.6% (26 of 57 participants; one additional lesion type: 31.6%; 2 additional lesion types: 14.0%); of these 26 participants with CT-occult intracranial lesions, 65.4% had axonal injury, 42.3% had subdural hematoma, and 23.1% had intracerebral contusion. GFAP levels were higher in participants with CT-occult MRI lesions compared with without (median: 630.6 pg/mL, Q1-Q3: [172.4-941.2] vs. 226.4 [105.8-436.1], p = 0.049), and were associated with axonal injury (no: median 226.7 pg/mL [109.6-435.1], yes: 828.6 pg/mL [204.0-1194.3], p = 0.009). Our results indicate that isolated tSAH on head CT is often not the sole intracranial traumatic injury in GCS 13-15 TBI. Forty-six percent of patients in our cohort (26 of 57 participants) had additional CT-occult traumatic lesions on MRI. Plasma GFAP may be an important biomarker for the identification of additional CT-occult injuries, including axonal injury. These findings should be interpreted cautiously given our small sample size and await validation from larger studies.

Performance of the IMPACT and CRASH prognostic models for traumatic brain injury in a contemporary multicenter cohort: a TRACK-TBI study.

OBJECTIVE: The International Mission on Prognosis and Analysis of Clinical Trials in Traumatic Brain Injury (IMPACT) and Corticosteroid Randomization After Significant Head Injury (CRASH) prognostic models for mortality and outcome after traumatic brain injury (TBI) were developed using data from 1984 to 2004. This study examined IMPACT and CRASH model performances in a contemporary cohort of US patients. METHODS: The prospective 18-center Transforming Research and Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) study (enrollment years 2014-2018) enrolled subjects aged ≥ 17 years who presented to level I trauma centers and received head CT within 24 hours of TBI. Data were extracted from the subjects who met the model criteria (for IMPACT, Glasgow Coma Scale [GCS] score 3-12 with 6-month Glasgow Outcome Scale-Extended [GOSE] data [n = 441]; for CRASH, GCS score 3-14 with 2-week mortality data and 6-month GOSE data [n = 831]). Analyses were conducted in the overall cohort and stratified on the basis of TBI severity (severe/moderate/mild TBI defined as GCS score 3-8/9-12/13-14), age (17-64 years or ≥ 65 years), and the 5 top enrolling sites. Unfavorable outcome was defined as GOSE score 1-4. Original IMPACT and CRASH model coefficients were applied, and model performances were assessed by calibration (intercept [< 0 indicated overprediction; > 0 indicated underprediction] and slope) and discrimination (c-statistic). RESULTS: Overall, the IMPACT models overpredicted mortality (intercept -0.79 [95% CI -1.05 to -0.53], slope 1.37 [1.05-1.69]) and acceptably predicted unfavorable outcome (intercept 0.07 [-0.14 to 0.29], slope 1.19 [0.96-1.42]), with good discrimination (c-statistics 0.84 and 0.83, respectively). The CRASH models overpredicted mortality (intercept -1.06 [-1.36 to -0.75], slope 0.96 [0.79-1.14]) and unfavorable outcome (intercept -0.60 [-0.78 to -0.41], slope 1.20 [1.03-1.37]), with good discrimination (c-statistics 0.92 and 0.88, respectively). IMPACT overpredicted mortality and acceptably predicted unfavorable outcome in the severe and moderate TBI subgroups, with good discrimination (c-statistic ≥ 0.81). CRASH overpredicted mortality in the severe and moderate TBI subgroups and acceptably predicted mortality in the mild TBI subgroup, with good discrimination (c-statistic ≥ 0.86); unfavorable outcome was overpredicted in the severe and mild TBI subgroups with adequate discrimination (c-statistic ≥ 0.78), whereas calibration was nonlinear in the moderate TBI subgroup. In subjects ≥ 65 years of age, the models performed variably (IMPACT-mortality, intercept 0.28, slope 0.68, and c-statistic 0.68; CRASH-unfavorable outcome, intercept -0.97, slope 1.32, and c-statistic 0.88; nonlinear calibration for IMPACT-unfavorable outcome and CRASH-mortality). Model performance differences were observed across the top enrolling sites for mortality and unfavorable outcome. CONCLUSIONS: The IMPACT and CRASH models adequately discriminated mortality and unfavorable outcome. Observed overestimations of mortality and unfavorable outcome underscore the need to update prognostic models to incorporate contemporary changes in TBI management and case-mix. Investigations to elucidate the relationships between increased survival, outcome, treatment intensity, and site-specific practices will be relevant to improve models in specific TBI subpopulations (e.g., older adults), which may benefit from the inclusion of blood-based biomarkers, neuroimaging features, and treatment data.

Malat1 fine-tunes bone homeostasis by orchestrating cellular crosstalk and the ß-catenin-OPG/Jagged1 pathway.

The IncRNA Malat1 was initially believed to be dispensable for physiology due to the lack of observable phenotypes in Malat1 knockout (KO) mice. However, our study challenges this conclusion. We found that both Malat1 KO and conditional KO mice in the osteoblast lineage exhibit significant osteoporosis. Mechanistically, Malat1 acts as an intrinsic regulator in osteoblasts to promote osteogenesis. Interestingly, Malat1 does not directly affect osteoclastogenesis but inhibits osteoclastogenesis in a non-autonomous manner in vivo via integrating crosstalk between multiple cell types, including osteoblasts, osteoclasts and chondrocytes. Our findings substantiate the existence of a novel remodeling network in which Malat1 serves as a central regulator by binding to ß-catenin and functioning through the ß-catenin-OPG/Jagged1 pathway in osteoblasts and chondrocytes. In pathological conditions, Malat1 significantly promotes bone regeneration in fracture healing. Bone homeostasis and regeneration are crucial to well-being. Our discoveries establish a previous unrecognized paradigm model of Malat1 function in the skeletal system, providing novel mechanistic insights into how a lncRNA integrates cellular crosstalk and molecular networks to fine tune tissue homeostasis, remodeling and repair.

Glioma-Immune Cell Crosstalk in Tumor Progression.

Glioma progression is a complex process controlled by molecular factors that coordinate the crosstalk between tumor cells and components of the tumor microenvironment (TME). Among these, immune cells play a critical role in cancer survival and progression. The complex interplay between cancer cells and the immune TME influences the outcome of immunotherapy and other anti-cancer therapies. Here, we present an updated view of the pro- and anti-tumor activities of the main myeloid and lymphocyte cell populations in the glioma TME. We review the underlying mechanisms involved in crosstalk between cancer cells and immune cells that enable gliomas to evade the immune system and co-opt these cells for tumor growth. Lastly, we discuss the current and experimental therapeutic options being developed to revert the immunosuppressive activity of the glioma TME. Knowledge of the complex interplay that elapses between tumor and immune cells may help develop new combination treatments able to overcome tumor immune evasion mechanisms and enhance response to immunotherapies.

VertebralArtery Injury with Anterior Cervical Spine Operations: A Systematic Review of Risk Factors, Clinical Outcomes, and Management Strategies.

OBJECTIVE: Anterior cervical spine operations are commonly performed on cervical spine pathologies and to a large extent are safe and successful. However, these surgical procedures expose the vertebral artery, posing a risk of harm to it. METHODS: A systematic review was conducted using PubMed, Google Scholar, and Web of Science electronic databases according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines to critically assess primary articles discussing treatment strategies "vertebral artery injury" AND "anterior cervical spine" and develop a management strategy based on our experience and meta-analysis of the literature. In addition, we present an illustrative case of iatrogenic vertebral artery injury presenting with 6 to 7 months' history of progressive dysphagia was transferred to our care from an outside institution. RESULTS: Included and analyzed were 43 articles that detailed 75 cases involving vertebral artery injury (VAI) in anterior cervical spine operations. Preoperatively, frequent clinical findings reported were sensory deficit (26 patients [63.41%]), motor deficit (20 patients [48.78%]), and pain (17 patients [41.46%]). In total, 32 patients (50.00%) endured injury of their left VA, and 30 patients had a right VAI. The 2 common causes of VAI were drilling (24 patients [40.00%]) and instrumentation (8 patients [13.33%]). CONCLUSIONS: Altogether, our review recommends repair or tamponade packing with a hemostatic agent for primary management. Should tamponade packing with a hemostatic agent be used for primary management, secondary management should entail either repair, stenting occlusion, embolization, anticoagulants, or ligation. Further examination of this treatment strategy based on a larger cohort is necessary.

Impact of obesity on complications and surgical outcomes after adult degenerative scoliosis spine surgery.

OBJECTIVE: To compare perioperative outcomes of obese versus non-obese adult patients who underwent degenerative scoliosis spine surgery. METHODS: 235 patients who underwent thoracolumbar adult spinal deformity (ASD) surgery (≥4 levels) were identified and categorized into two cohorts based on their body mass indices (BMI): obese (BMI ≥30 kg/m2; n = 81) and non-obese (BMI <30 kg/m2; n = 154). Preoperative (demographics, co-morbidities, American Society of Anesthesiologists (ASA) score and modified frailty indices (mFI-5 and mFI-11)), intraoperative (estimated blood loss (EBL) and anesthesia duration), and postoperative (complication rates, Oswestry Disability Index (ODI) scores, discharge destination, readmission rates, and survival) characteristics were analyzed by student's t, chi-squared, and Mann-Whitney U tests. RESULTS: Obese patients were more likely to be Black/African-American (p < 0.05, OR:4.11, 95% CI:1.20-14.10), diabetic (p < 0.05, OR:10.18, 95% CI:4.38-23.68) and had higher ASA (p < .01) and psoas muscle indices (p < 0.0001). Furthermore, they had greater pre- and post-operative ODI scores (p < 0.05) with elevated mFI-5 (p < 0.0001) and mFI-11 (p < 0.01). Intraoperatively, obese patients were under anesthesia for longer time periods (p < 0.05) with higher EBL (p < 0.05). Postoperatively, while they were more likely to have complications (OR:1.77, 95% CI:1.01 - 3.08), had increased postop days to initiate walking (p < .05) and were less likely to be discharged home (OR:0.55, 95% CI:0.31-0.99), no differences were found in change in ODI scores or readmission rates between the two cohorts. CONCLUSIONS: Obesity increases pre-operative risk factors including ASA, frailty and co-morbidities leading to longer operations, increased EBL, higher complications and decreased discharge to home. Pre-operative assessment and systematic measures should be taken to improve peri-operative outcomes.

Transfusion guidelines in adult spine surgery: a systematic review and critical summary of currently available evidence.

BACKGROUND CONTEXT: Red blood cell transfusion can be associated with complications in medical and surgical patients. Acute anemia in ambulatory patients undergoing surgery can also impede wound healing and independent self-care. Current transfusion threshold guidelines are still based on evidence derived from critically-ill intensive care unit medical patients and may not apply to spine surgery candidates. PURPOSE: We aimed to provide the reader with a synthesis of the best available evidence to recommend transfusion trigger thresholds and guidelines in adult patients undergoing spine surgery. STUDY DESIGN/SETTING: This is a systematic review. OUTCOME MEASURES: Physiological measure: Blood transfusion thresholds and associated posttransfusion complications (morbidity, mortality, length of stay, infections, etc) of the published articles. PATIENT SAMPLE: Adult spine surgery patients. METHODS: A systematic review of the literature using the PubMed, Google Scholar, and Web of Science electronic databases was made according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Focus was set on papers discussing thresholds for blood transfusion in adult surgical spine patients, as well as complications associated with transfusion after acute surgical blood loss in the operating room or postoperative period. Publications discussing pediatric cases, blood type analyses, blood loss prevention strategies and protocols, systematic reviews and letters to the editor were excluded. RESULTS: A total of 22 articles fitting our search criteria were reviewed. Patients who received blood transfusion in these studies were older, of female gender, had more severe comorbidities except for smoking, and had prolonged surgical time. Blood transfusion was associated with multiple adverse postoperative complications, including a higher rate of superficial or deep surgical site infections, sepsis, urinary and pulmonary infections, cardiovascular complications, return to the operating room, and increased postoperative length of stay and 30 day readmission. Analysis of transfusion thresholds from these studies showed that a pre-operative hemoglobin (Hb) of > 13 g/dL, and an intraoperative and post-operative Hb nadir above 9 and 8 g/dL, respectively, were associated with better outcomes and fewer wound infections than lower thresholds (Level B Class III). Additionally, it was generally recommended to transfuse autologous blood that was < 28 days old, if possible, with a limit of 2 to 3 units to minimize patient morbidity and mortality. CONCLUSIONS: Blood transfusion thresholds in surgical patients may be specialty-specific and different than those used for critically-ill medical patients. For adult spine surgery patients, red blood cell transfusion should be avoided if Hb numbers remain > 9 and 8 g/dL in the intraoperative and direct post-operative periods, respectively.

NORAD-induced Pumilio phase separation is required for genome stability.

Liquid-liquid phase separation is a major mechanism of subcellular compartmentalization1,2. Although the segregation of RNA into phase-separated condensates broadly affects RNA metabolism3,4, whether and how specific RNAs use phase separation to regulate interacting factors such as RNA-binding proteins (RBPs), and the phenotypic consequences of such regulatory interactions, are poorly understood. Here we show that RNA-driven phase separation is a key mechanism through which a long noncoding RNA (lncRNA) controls the activity of RBPs and maintains genomic stability in mammalian cells. The lncRNA NORAD prevents aberrant mitosis by inhibiting Pumilio (PUM) proteins5-8. We show that NORAD can out-compete thousands of other PUM-binding transcripts to inhibit PUM by nucleating the formation of phase-separated PUM condensates, termed NP bodies. Dual mechanisms of PUM recruitment, involving multivalent PUM-NORAD and PUM-PUM interactions, enable NORAD to competitively sequester a super-stoichiometric amount of PUM in NP bodies. Disruption of NORAD-driven PUM phase separation leads to PUM hyperactivity and genome instability that is rescued by synthetic RNAs that induce the formation of PUM condensates. These results reveal a mechanism by which RNA-driven phase separation can regulate RBP activity and identify an essential role for this process in genome maintenance. The repetitive sequence architecture of NORAD and other lncRNAs9-11 suggests that phase separation may be a widely used mechanism of lncRNA-mediated regulation.

Compressive Cervical Myelopathy in Patients With Demyelinating Disease of the Central Nervous System: Improvement After Surgery Despite a Late Diagnosis.

Objective We aimed to assess the impact of surgical intervention on outcome in patients diagnosed with demyelinating disorders and cervical degenerative disease warranting surgical intervention. Methods The records of patients with a diagnosis of a demyelinating disorder of the central nervous system who underwent cervical spine surgery at a single institution from 2016 to 2020 were reviewed. Demyelinating disease included multiple sclerosis (MS), neuromyelitis optica, and transverse myelitis (TM). The dates of initial spine symptom onset, recognition of spinal pathology by the primary provider, referral to spine surgery, and spine surgery procedures were collected. Hospital length of stay (LOS) and postoperative outcomes and complications were recorded. Results A total of 19 patients with a diagnosis of demyelinating disorders underwent cervical spine surgery at our institution. Seventeen patients had MS. The average time interval between a documented diagnosis of myelopathy or radiculopathy and referral to the Spine clinic was 67.95 months (M=40, SD=64.87). Twelve patients had imaging studies depicting degenerative spine disease that would warrant surgical intervention at the time of examination by their primary physician. The average delay for referral to the Spine clinic for these patients was 16.5 months (M=5; SD=25.36). More than 89% of patients experienced significant neurologic improvement postoperatively. Conclusions There is a delay in the recognition of cervical spine disease amenable to a surgical resolution in patients with demyelinating disorders. Surgical treatment can lead to significant clinical improvement in this patient population even if delayed, and likely carries similar risk to that of the general population.

Intramedullary Spinal Metastatic Renal Cell Carcinoma: Systematic Review of Disease Presentation, Treatment, and Prognosis with Case Illustration.

OBJECTIVE: Renal cell carcinoma (RCC) metastases to the intramedullary spinal cord carry a grim prognosis. The purpose of this review is to provide the reader with a comprehensive and systematic review of the current literature, and to present an illustrative case that would aid in the future management of similar scenarios. METHODS: A systematic review of the literature using the PubMed electronic database was made according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Only human clinical reports of intramedullary RCC metastasis were included. We also present an illustrative case that was treated at our institution. RESULTS: We identified 23 reports with a total of 31 patients. Of the tumors, 47% were located at the cervical level. Brain metastases were present in 41% of cases. Limb weakness (72%), urinary incontinence (41%), dysesthesia (47%), and localized spinal pain (38%) were the most frequently reported symptoms. Surgical resection alone was used in 34% of cases, followed by a combination of surgery and radiotherapy (31%), and radiotherapy alone (25%). Spinal metastases were detected an average of 32.1 months after the diagnosis of RCC, and mean patient survival after that was 8 months (range, 0-65 months). Reported survival after radiotherapy appeared to be the longest (11.2 months) compared with surgery (9.1 months) and combination therapy (5 months). CONCLUSIONS: Intramedullary spinal metastatic RCC is a rare entity with debilitating neurologic potential. Survival appears to be affected by the treatment method but is also likely influenced by the stage of discovery of the disease.

PUMILIO, but not RBMX, binding is required for regulation of genomic stability by noncoding RNA NORAD.

NORAD is a conserved long noncoding RNA (lncRNA) that is required for genome stability in mammals. NORAD acts as a negative regulator of PUMILIO (PUM) proteins in the cytoplasm, and we previously showed that loss of NORAD or PUM hyperactivity results in genome instability and premature aging in mice (Kopp et al., 2019). Recently, however, it was reported that NORAD regulates genome stability through an interaction with the RNA binding protein RBMX in the nucleus. Here, we addressed the contributions of NORAD:PUM and NORAD:RBMX interactions to genome maintenance by this lncRNA in human cells. Extensive RNA FISH and fractionation experiments established that NORAD localizes predominantly to the cytoplasm with or without DNA damage. Moreover, genetic rescue experiments demonstrated that PUM binding is required for maintenance of genomic stability by NORAD whereas binding of RBMX is dispensable for this function. These data provide an important foundation for further mechanistic dissection of the NORAD-PUMILIO axis in genome maintenance.

PUMILIO hyperactivity drives premature aging of Norad-deficient mice.

Although numerous long noncoding RNAs (lncRNAs) have been identified, our understanding of their roles in mammalian physiology remains limited. Here, we investigated the physiologic function of the conserved lncRNA Norad in vivo. Deletion of Norad in mice results in genomic instability and mitochondrial dysfunction, leading to a dramatic multi-system degenerative phenotype resembling premature aging. Loss of tissue homeostasis in Norad-deficient animals is attributable to augmented activity of PUMILIO proteins, which act as post-transcriptional repressors of target mRNAs to which they bind. Norad is the preferred RNA target of PUMILIO2 (PUM2) in mouse tissues and, upon loss of Norad, PUM2 hyperactively represses key genes required for mitosis and mitochondrial function. Accordingly, enforced Pum2 expression fully phenocopies Norad deletion, resulting in rapid-onset aging-associated phenotypes. These findings provide new insights and open new lines of investigation into the roles of noncoding RNAs and RNA binding proteins in normal physiology and aging.

Four-Level Vertebrectomy for En Bloc Resection of a Cervical Chordoma.

BACKGROUND: Chordomas are locally aggressive tumors that can involve multiple levels of the spine and are difficult to resect. We present our technique for 4-level en bloc cervical spondylectomy for a locally aggressive chordoma. CASE DESCRIPTION: A 37-year-old woman presented with a 6-month history of dysphagia and a large indurated cervical mass. Imaging showed an enhancing lesion involving C3-6. Needle biopsy confirmed the diagnosis of chordoma. En bloc resection was chosen to maximize her chances of disease-free survival. A 360° approach was deemed necessary. We posteriorly disconnected the vertebral bodies and skeletonized the bilateral vertebral arteries and nerve roots. The interspinous and yellow ligaments and the spinous processes were spared to maintain a solid posterior tension band, as previously described approaches that had sacrificed these elements had a high rate of instrumentation failure. After posterior instrumentation, a wide anterior approach enabled us to resect the tumor attached to the vertebral bodies of C3-6 as 1 specimen. A 4-level corpectomy cage and plate were used for anterior instrumentation. The patient tolerated the surgery well. She needed a temporary gastrostomy, and she had a right C5 palsy that progressively recovered. Follow-up imaging showed no tumor recurrence and good bony fusion. CONCLUSIONS: En bloc resection as part of a multidisciplinary team approach remains the mainstay of spinal chordoma treatment. Modern instrumentation and careful dissection can provide good results even in locally advanced cases.

RBP-J-Regulated miR-182 Promotes TNF-α-Induced Osteoclastogenesis.

Increased osteoclastogenesis is responsible for osteolysis, which is a severe consequence of inflammatory diseases associated with bone destruction, such as rheumatoid arthritis and periodontitis. The mechanisms that limit osteoclastogenesis under inflammatory conditions are largely unknown. We previously identified transcription factor RBP-J as a key negative regulator that restrains TNF-α-induced osteoclastogenesis and inflammatory bone resorption. In this study, we tested whether RBP-J suppresses inflammatory osteoclastogenesis by regulating the expression of microRNAs (miRNAs) important for this process. Using high-throughput sequencing of miRNAs, we obtained the first, to our knowledge, genome-wide profile of miRNA expression induced by TNF-α in mouse bone marrow-derived macrophages/osteoclast precursors during inflammatory osteoclastogenesis. Furthermore, we identified miR-182 as a novel miRNA that promotes inflammatory osteoclastogenesis driven by TNF-α and whose expression is suppressed by RBP-J. Downregulation of miR-182 dramatically suppressed the enhanced osteoclastogenesis program induced by TNF-α in RBP-J-deficient cells. Complementary loss- and gain-of-function approaches showed that miR-182 is a positive regulator of osteoclastogenic transcription factors NFATc1 and B lymphocyte-induced maturation protein-1. Moreover, we identified that direct miR-182 targets, Foxo3 and Maml1, play important inhibitory roles in TNF-α-mediated osteoclastogenesis. Thus, RBP-J-regulated miR-182 promotes TNF-α-induced osteoclastogenesis via inhibition of Foxo3 and Maml1. Suppression of miR-182 by RBP-J serves as an important mechanism that restrains TNF-α-induced osteoclastogenesis. Our results provide a novel miRNA-mediated mechanism by which RBP-J inhibits osteoclastogenesis and suggest that targeting of the newly described RBP-J-miR-182-Foxo3/Maml1 axis may represent an effective therapeutic approach to suppress inflammatory osteoclastogenesis and bone resorption.

Roles of subunit NuoL in the proton pumping coupling mechanism of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli.

Respiratory complex I has an L-shaped structure formed by the hydrophilic arm responsible for electron transfer and the membrane arm that contains protons pumping machinery. Here, to gain mechanistic insights into the role of subunit NuoL, we investigated the effects of Mg2+, Zn2+ and the Na+/H+ antiporter inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on proton pumping activities of various isolated NuoL mutant complex I after reconstitution into Escherichia coli double knockout (DKO) membrane vesicles lacking complex I and the NADH dehydrogenase type 2. We found that Mg2+ was critical for proton pumping activity of complex I. At 2 µM Zn2+, proton pumping of the wild-type was selectively inhibited without affecting electron transfer; no inhibition in proton pumping of D178N and D400A was observed, suggesting the involvement of these residues in Zn2+ binding. Fifteen micromolar of EIPA caused up to ∼40% decrease in the proton pumping activity of the wild-type, D303A and D400A/E, whereas no significant change was detected in D178N, indicating its possible involvement in the EIPA binding. Furthermore, when menaquinone-rich DKO membranes were used, the proton pumping efficiency in the wild-type was decreased significantly (∼50%) compared with NuoL mutants strongly suggesting that NuoL is involved in the high efficiency pumping mechanism in complex I.

Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2).

Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O2 activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC50 = ∼1 µm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O2 activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O2 activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells.

Semiquinone intermediates are involved in the energy coupling mechanism of E. coli complex I.

Complex I (NADH:quinone oxidoreductase) is central to cellular aerobic energy metabolism, and its deficiency is involved in many human mitochondrial diseases. Complex I translocates protons across the membrane using electron transfer energy. Semiquinone (SQ) intermediates appearing during catalysis are suggested to be key for the coupling mechanism in complex I. However, the existence of SQ has remained controversial due to the extreme difficulty in detecting unstable and low intensity SQ signals. Here, for the first time with Escherichia coli complex I reconstituted in proteoliposomes, we successfully resolved and characterized three distinct SQ species by EPR. These species include: fast-relaxing SQ (SQNf) with P1/2 (half-saturation power level)>50mW and a wider linewidth (12.8 G); slow-relaxing SQ (SQNs) with P1/2=2-3mW and a 10G linewidth; and very slow-relaxing SQ (SQNvs) with P1/2= ~0.1mW and a 7.5G linewidth. The SQNf signals completely disappeared in the presence of the uncoupler gramicidin D or squamotacin, a potent E. coli complex I inhibitor. The pH dependency of the SQNf signals correlated with the proton-pumping activities of complex I. The SQNs signals were insensitive to gramicidin D, but sensitive to squamotacin. The SQNvs signals were insensitive to both gramicidin D and squamotacin. Our deuterium exchange experiments suggested that SQNf is neutral, while SQNs and SQNvs are anion radicals. The SQNs signals were lost in the ΔNuoL mutant missing transporter module subunits NuoL and NuoM. The roles and relationships of the SQ intermediates in the coupling mechanism are discussed.

Semiquinone and cluster N6 signals in His-tagged proton-translocating NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli.

NADH:ubiquinone oxidoreductase (complex I) pumps protons across the membrane using downhill redox energy. The Escherichia coli complex I consists of 13 different subunits named NuoA-N coded by the nuo operon. Due to the low abundance of the protein and some difficulty with the genetic manipulation of its large ~15-kb operon, purification of E. coli complex I has been technically challenging. Here, we generated a new strain in which a polyhistidine sequence was inserted upstream of nuoE in the operon. This allowed us to prepare large amounts of highly pure and active complex I by efficient affinity purification. The purified complex I contained 0.94 ± 0.1 mol of FMN, 29.0 ± 0.37 mol of iron, and 1.99 ± 0.07 mol of ubiquinone/1 mol of complex I. The extinction coefficient of isolated complex I was 495 mM(-1) cm(-1) at 274 nm and 50.3 mM(-1) cm(-1) at 410 nm. NADH:ferricyanide activity was 219 ± 9.7 µmol/min/mg by using HEPES-Bis-Tris propane, pH 7.5. Detailed EPR analyses revealed two additional iron-sulfur cluster signals, N6a and N6b, in addition to previously assigned signals. Furthermore, we found small but significant semiquinone signal(s), which have been reported only for bovine complex I. The line width was ~12 G, indicating its neutral semiquinone form. More than 90% of the semiquinone signal originated from the single entity with P½ (half-saturation power level) = 1.85 milliwatts. The semiquinone signal(s) decreased by 60% when with asimicin, a potent complex I inhibitor. The functional role of semiquinone and the EPR assignment of clusters N6a/N6b are discussed.