Anion-accelerated asymmetric Nazarov cyclization: access to vicinal all-carbon quaternary stereocenters.

We have developed a catalytic asymmetric Nazarov cyclization that results in the formation of two contiguous all-carbon quaternary stereocenters in high yield with excellent levels of asymmetric induction. This method requires no catalyst recognition elements in the starting materials that are simple diketoesters. Geometrically pure E or Z isomers of the starting material lead to diastereomerically pure products with high enantioselectivity because the species that undergoes cyclization is a rhodium enolate that is configurationally stable.

An Inexpensive, Multimodal Simulation Model for Teaching Ultrasound Identification of Soft Tissue Pathology and Regional Anesthesia.

Ultrasound identification of soft tissue pathology is a useful skill for the emergency physician, but it requires practice and familiarity to be effective. Given its rising popularity in the Emergency Department, regional anesthesia is another essential skill that requires practice. Realistic models can help create procedural confidence and accuracy. Since entry-level professional-grade models can be cost-prohibitive, the development of simple and affordable models for teaching is valuable for emergency provider education, especially in resource-limited settings. Other inexpensive models have been produced and discussed in ultrasound; literature; however, no models have yet been designed for the replication of several different modalities in a single model. We developed and successfully tested a meat phantom model utilizing materials available at a local grocery store that can be quickly assembled in a short amount of time with minimal effort. This low-cost, easy-to-make phantom accurately replicates human tissue and pathology and is ideal for learners to practice several skill sets at once.

Natural product preferentially targets redox and metabolic adaptations and aberrantly active STAT3 to inhibit breast tumor growth in vivo.

Dysregulated gene expression programs and redox and metabolic adaptations allow cancer cells to survive under high oxidative burden. These mechanisms also represent therapeutic vulnerabilities. Using triple-negative breast cancer (TNBC) as a model, we show that compared to normal human breast epithelial cells, the TNBC cells, MDA-MB-231 and MDA-MB-468 that harbor constitutively active STAT3 also express higher glucose-6-phosphate dehydrogenase (G6PD), thioredoxin reductase (TrxR)1, NADPH, and GSH levels for survival. Present studies discover that the natural product, R001, targets these adaptation mechanisms. Treatment of TNBC cells with R001 inhibited constitutively active STAT3, STAT3-regulated gene expression, and the functions of G6PD and TrxR1. Consequently, in the TNBC, but not normal cells, R001 suppressed GSH levels, but raised NADPH levels, reflective of a loss of mitochondrial respiration and which led to reactive oxygen species (ROS) induction, all of which led to loss of viable cells and inhibition of anchorage-dependent and independent growth. R001 treatment further led to early pyroptosis and late DNA damage, cell cycle arrest, and apoptosis only in the TNBC cells. Oral administration of 5 mg/kg R001 inhibited MDA-MB-468 xenografts growth in mice, with reduced pY705-STAT3, G6PD, TrxR1, and GSH levels. R001 serves as a therapeutic entity that targets the vulnerabilities of TNBC cells to inhibit tumor growth in vivo.

Fluorine on fluorenes.

A practical synthesis of 1,8-difluorofluorenone and of its (2,7)-dibromo and (2,4,5,7)-tetrabromo derivatives has been developed. Fluorinated fluorenones are the starting materials for polyhalogenated dibenzochrysenes as well as for geodesic hydrocarbons. 2,7-Dibromo-1,8-difluoro-9H-fluoren-9-one was converted to 3,14-dibromo-4,13-difluorodibenzo[g,p]chrysene through a novel strategy.

Mycobacterium marinum cellulitis: A fishy etiology.

Skin and soft tissue infections account for 2% of emergency department visits annually, though more unusual causative bacteria associated with saltwater exposure may result in morbidity. Mycobacterium marinum represents a rare but important cause of cellulitis, which if untreated or improperly managed, can progress to dactylitis or osteomyelitis. This unusual diagnosis is made more challenging due to the prolonged incubation period of approximately 21 days, temporally separating the inoculation from the disease. Patients will present with a nodular rash in a sporotrichoid pattern. While doxycycline is one antibiotic providing saltwater coverage, M. marinum has variable sensitivities to anti-mycobacterial antibiotics, and thus biopsy helps confirm the diagnosis as well as provide sensitivities for treatment. Emergency clinicians should inquire about environmental risk factors when caring for patients with cellulitis, especially with atypical skin presentations, and consider M. marinum as a rare but important cellulitis etiology.

Modular Synthesis of a Semibuckminsterfullerene.

A convergent synthesis of dibenzochrysenes and diindenochrysenes that proceeds from difluorofluorenes and acetoxyenone 15 has been used to prepare 5,6,11,12-tetrabromosemibuckminsterfullerene (31). The synthesis is highly modular and is distinguished by proceeding through an unsymmetrical intermediate. Our work will enable the straightforward preparation of semibuckminsterfullerenes from diindenochrysenes that lack bilateral symmetry using common reagents and nonpyrolytic conditions.

Tattoo Associated Native Valve Infective Endocarditis.

Infective Endocarditis (IE) is an uncommon illness in the emergency department (ED) with significant associated morbidity. Patients with IE typically possess risk factors predisposing them to bacterial invasion. These risk factors include intravenous drug use, valvulopathies, valve replacement, poor oral hygiene, immunocompromised state, and recent invasive procedures. A rarer condition is tattoo-associated IE. Diagnosis of IE includes multiple blood cultures and echocardiography. Therapeutic interventions include prolonged intravenous antibiotics and potential surgery. Complications from IE can be severe, including organ injury from septic emboli, heart failure, valvular insufficiency, bacteremia, and fulminant septic shock. Prompt identification and treatment are necessary to reduce patient morbidity and mortality.

Synthesis of Fluorenes and Dibenzo[g,p]chrysenes through an Oxidative Cascade.

We have developed robust, operationally simple syntheses of fluorenes and of dibenzo[g,p]chrysenes through oxidative cascade processes. These structures that are commonly encountered in optoelectronic materials, dyes, and pharmaceutical products are accessible from 1,4-dioxaspiro[4.5]decan-8-one. The reactions are conducted open to air with inexpensive, safe CuBr2 or CuCl2.

Small Bowel in Vagina: A Case for Pelvic Exams.

Vaginal cuff dehiscence with small bowel evisceration is a serious but uncommon postoperative complication of total laparoscopic hysterectomies. The severity of surgical site dehiscence can range from small, partial to full-thickness wound dehiscence, manifesting with acute abdominal pain, vaginal bleeding, or discharge, and is often precipitated by sexual intercourse. While imaging, including a pelvic ultrasound and computed tomography (CT), may help in undifferentiated acute abdominal pain, vaginal cuff dehiscence remains a clinical diagnosis found on physical exam. Because vaginal cuff dehiscence is a clinical diagnosis, sparse data exist regarding radiographic sensitivities and specificities in the identification of vaginal cuff dehiscence. Despite the increasing literature suggesting that pelvic exams are invasive with often limited utility, the authors argue that pelvic exams remain essential in identifying complications of hysterectomies. The authors present a case of a 40-year-old woman with acute abdominal pain found to have loops of small bowel in the vaginal vault, discovered only on physical exam after negative CT and ultrasound imaging.

Third-trimester growth diversity in small fetuses classified as appropriate-for-gestational age or small-for-gestational age at birth.

OBJECTIVE: We have shown previously that third-trimester growth in small fetuses (estimated fetal weight (EFW) < 10th percentile) with birth weight (BW) < 10th percentile is heterogeneous using individualized growth assessment (IGA). We aimed to test our hypothesis that individual growth patterns in small fetuses with BW > 10th percentile are also variable but in different ways. METHODS: This was a study of 191 cases with EFW < 10th percentile and BW > 10th percentile (appropriate-for-gestational-age (AGA) cohort), derived from the PORTO study. Composite size parameters were used to quantify growth pathology at individual third-trimester timepoints (individual composite prenatal growth assessment score (-icPGAS)). The fetal growth pathology score 1 (-FGPS1), calculated cumulatively from serial -icPGAS values, was used to characterize third-trimester growth patterns. Vascular-system evaluation included umbilical artery (UA) and middle cerebral artery (MCA) Doppler velocimetry. Outcome variables were birth age (preterm/term delivery) and BW (expressed as growth potential realization index for weight (GPRIWT ) and percentile). The findings from the AGA cohort were compared with those from small fetuses (EFW < 10th percentile) with BW < 10th percentile (small-for-gestational-age (SGA) cohort). RESULTS: The AGA cohort was found to have 134 fetuses (70%) with normal growth pattern and 57 (30%) with growth restriction based on IGA criteria. Seven growth-restriction -FGPS1 patterns were observed, including the previously defined progressive, late, adaptive and recovering types. The recovering type was the most common growth pattern in the AGA cohort (50.9%). About one-third of fetuses without any evidence of growth restriction had significant unexplained abnormalities in the UA (34%) and MCA (31%) and elevated mean GPRIWT values (113 ± 12.5%). Comparison of the AGA and SGA cohorts indicated a significant difference in the distribution of -FGPS1 growth patterns (P = 0.0001). Compared with the SGA cohort, the AGA cohort had more fetuses with a normal growth pattern (70% vs 38%) and fewer cases with growth restriction (30% vs 62%). While the recovering type was the most common growth-restriction pattern in the AGA cohort (51%), the progressive type was the primary growth-restriction pattern in the SGA cohort (44%). No difference in the incidence of MCA or UA abnormality was found between the SGA and AGA cohorts when comparing subgroups of more than 10 fetuses. CONCLUSIONS: Both normal-growth and growth-restriction patterns were observed in the AGA cohort using IGA, as seen previously in the SGA cohort. The seven types of growth restriction defined in the SGA cohort were also identified in AGA cases, but their distribution was significantly different. In one-third of cases without evidence of growth pathology in the AGA cohort, Doppler abnormalities in the UA and MCA were seen. This heterogeneity underscores the difficulty of accurate classification of fetal and neonatal growth status using conventional population-based methods. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Leveraging the Existing Anesthesia Information Management System to Improve Anesthesia Quality Assurance Outcome Reporting.

INTRODUCTION: Tracking measures of quality over time has been shown to improve care within institutions and across health systems. Perioperative quality assurance (QA) tracking by anesthesia departments in the Military Health System (MHS) has not used a uniform system integrated into the workflow of anesthesia providers. The purpose of this study was to demonstrate that the use of the embedded QA outcome reporting feature in the anesthesia information management system (AIMS) increased the rate of reporting compared to the current paper reporting system in a military anesthesia department. MATERIALS AND METHODS: An electronic outcome reporting mechanism embedded in the AIMS was activated as an alternative to paper QA outcome reporting. The proportion of anesthesia cases per month in a 12-month period with a reported QA outcome was compared to the previous year in which only the paper reporting system was used. The total number of cases in each time period with an outcome reported was compared using chi square for proportions, and systems were evaluated using the Statistical Process Control methodology. This project was evaluated and determined to be exempt from review by our institutional review board. RESULTS: There was a 389.8% increase in the number of cases with a QA outcome reported after the implementation of the outcome reporting function integrated into the AIMS (χ2 = 207.72; P <.001, Table I). Systems before and after the intervention were stable, and special cause variation was noted only at the point of implementation of the electronic reporting system. Anesthesia providers were surveyed and felt that the addition of QA reporting to the AIMS made QA reporting more likely. CONCLUSIONS: The use of an electronic QA outcome reporting method integrated into the AIMS dramatically increased the likelihood that a QA outcome would be reported. The decreased administrative burden of the integrated outcome reporting system was likely the primary reason for this increase. This study was limited by the fact that it was done in a single institution; however, the size and timing of the increase clearly indicate that the intervention was the reason for improved reporting. Electronic health record upgrades should consider incorporating QA reporting into the AIMS across the MHS. These measures could allow for system-wide improvement, evaluation, and evidence-based education on their own, but also by facilitating participation in the American Society of Anesthesiologists' Anesthesia Quality Institute's National Anesthesia Clinical Outcomes Registry. This report serves as a valuable example to institutions and perioperative leaders in the MHS of how to improve the robustness of perioperative QA reporting such that it could be used to validate and improve the value of care.

Can char carbon enhance soil properties and crop yields in low-carbon soils?

Restoring soil carbon (C) lost due to intensive farming is a long-term endeavor under current conservation management practices. Application of coal combustion residue (293 g C kg-1 ) from a sugar beet (Beta vulgaris L.) processing factory, hereafter referred to as char, could rapidly restore soil C and productivity in degraded croplands, but data on this potential strategy are unavailable. We assessed the impacts of char application to two relatively low-C soils (10.1 and 12.2 g C kg-1 ) and one relatively high-C soil (17.3 g C kg-1 ) on soil C, soil physical and fertility properties, and crop yields in no-till systems in the Great Plains after 2 yr. Char was disked to 15 cm soil depth at char-C application rates ranging from 0 to 19.7 Mg C ha-1 , corresponding to char application rates ranging from 0 to 67.3 Mg ha-1 . The highest char rate increased C concentration in all soils but increased C stock only in low-C soils. Char did not affect soil penetration resistance, available water, aggregate stability, most nutrients, and crop yields. Char application at high rates increased sulfate, Ca, Mg, and Na concentrations but did not influence other properties. Carbon recovery of the char applied at the highest rate varied among soils from 50 to 85%, but the mechanisms for such differences need further investigation. Short-term duration, low char C concentration, and low application rates may explain the limited char effects. Overall, char application at 19.7 Mg char-C ha-1 (i.e., 67.3 Mg char ha-1 ) increased soil C concentration but had negligible effects on other soil properties and crop yields after 2 yr.

Sonographic markers of fetal adiposity and risk of Cesarean delivery.

OBJECTIVE: Increased fetal size is associated with shoulder dystocia during labor and subsequent need for assisted delivery. We sought to investigate if increased fetal adiposity diagnosed sonographically in late pregnancy is associated with increased risk of operative delivery. METHODS: This secondary analysis of the Genesis Study recruited 2392 nulliparous women with singleton pregnancy in cephalic presentation, in a prospective, multicenter study, to examine prenatal and intrapartum predictors of Cesarean delivery. Participants underwent ultrasound and clinical evaluation between 39 + 0 and 40 + 6 weeks' gestation. Data on fetal biometry were not revealed to patients or to their managing clinicians. A fetal adiposity composite of fetal thigh adiposity and fetal abdominal wall thickness was compiled for each infant in order to determine whether fetal adiposity > 90th centile was associated with an increased risk of Cesarean or operative vaginal delivery. RESULTS: After exclusions, data were available for 2330 patients. Patients with a fetal adiposity composite > 90th centile had a higher maternal body mass index (BMI) (25 ± 5 kg/m2 vs 24 ± 4 kg/m2 ; P = 0.005), birth weight (3872 ± 417 g vs 3585 ± 401 g; P < 0.0001) and rate of induction of labor (47% (108/232) vs 40% (834/2098); P = 0.048) than did those with an adiposity composite ≤ 90th centile. Fetuses with adiposity composite > 90th centile were more likely to require Cesarean delivery than were those with adiposity composite ≤ 90th centile (P < 0.0001). After adjusting for birth weight, maternal BMI and need for induction of labor, fetal adiposity > 90th centile remained a risk factor for Cesarean delivery (P < 0.0001). A fetal adiposity composite > 90th centile was more predictive of the need for unplanned Cesarean delivery than was an estimated fetal weight > 90th centile (odds ratio, 2.20 (95% CI, 1.65-2.94; P < 0.001) vs 1.74 (95% CI, 1.29-2.35; P < 0.001). Having an adiposity composite > 90th centile was not associated with an increased likelihood of operative vaginal delivery when compared with having an adiposity composite ≤ 90th centile (P = 0.37). CONCLUSIONS: Fetuses with increased adipose deposition are more likely to require Cesarean delivery than are those without increased adiposity. Consideration should, therefore, be given to adding fetal thigh adiposity and abdominal wall thickness to fetal sonographic assessment in late pregnancy. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Pannexin 1 regulates adipose stromal cell differentiation and fat accumulation.

Pannexin 1 (Panx1) is a channel-forming glycoprotein important in paracrine signaling and cellular development. In this study, we discovered that mice globally lacking Panx1 (KO) have significantly greater total fat mass and reduced lean mass compared to wild type (WT) mice under a normal diet. Despite having higher fat content, Panx1 KO mice on a high fat diet exhibited no differences in weight gain and blood markers of obesity as compared to WT controls, except for an increase in glucose and insulin levels. However, metabolic cage data revealed that these Panx1 KO mice display significantly increased activity levels, higher ambulatory activity, and reduced sleep duration relative to their WT littermates on a high-fat diet. To uncover the cellular mechanism responsible for the increased fat content in the KO, we isolated primary cultures of adipose-derived stromal cells (ASCs) from WT and KO fat pads. In WT ASCs we observed that Panx1 protein levels increase upon induction into an adipogenic lineage. ASCs isolated from Panx1 KO mice proliferate less but demonstrate enhanced adipogenic differentiation with increased intracellular lipid accumulation, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and adipokine secretion, as compared to WT ASCs. This was consistent with the increased adipocyte size and decreased adipocyte numbers observed in subcutaneous fat of the Panx1 KO mice compared to WT. We concluded that Panx1 plays a key role in adipose stromal cells during the early stages of adipogenic proliferation and differentiation, regulating fat accumulation in vivo.

Antitumor Effects of DC Vaccine With ALA-PDT-Induced Immunogenic Apoptotic Cells for Skin Squamous Cell Carcinoma in Mice.

Targeted immunotherapy using dendritic cell vaccine has been employed for the treatment of solid tumors. Topical 5-aminolevulinic acid-mediated photodynamic therapy, an established approach for topical cancers, can induce an effective antitumor immune response. We have previously shown that 5-aminolevulinic acid-mediated photodynamic therapy-induced tumor lysates could considerably enhance antigen-presenting capacity of ex vivo-generated dendritic cells. The current study further demonstrates that 5-aminolevulinic acid-mediated photodynamic therapy dendritic cell vaccine can induce immune responses against cancers. Dendritic cells pulsed by photodynamic therapy-treated skin squamous cell carcinoma cells inhibited squamous cell carcinoma to a greater extent than tumor lysates treated by photodynamic therapy alone or dendritic cells pulsed by freeze-thawed treated tumor cells. Immunohistochemistry showed that photodynamic therapy dendritic cell vaccine could increase the activity of CD4+ and CD8+ T cells in the tumor implantation sites. Flow cytometry assays showed that CD4+ and CD8+ T cells in the spleens of photodynamic therapy dendritic cell vaccine immunized mice increased significantly. Furthermore, we observed increased amounts of interleukin 12 and Interferon gamma (IFN-γ) and decreased amounts of interleukin 10 in the splenocytes and peripheral blood of photodynamic therapy dendritic cell vaccine immunized mice by enzyme linked immunosorbent assay (ELISA). Taken together, our findings suggest that photodynamic therapy dendritic cell vaccination is an effective prophylactic therapy for squamous cell carcinoma.

Collagenase treatment enhances proteomic coverage of low-abundance proteins in decellularized matrix bioscaffolds.

There is great interest in the application of advanced proteomic techniques to characterize decellularized tissues in order to develop a deeper understanding of the effects of the complex extracellular matrix (ECM) composition on the cellular response to these pro-regenerative bioscaffolds. However, the identification of proteins in ECM-derived bioscaffolds is hindered by the high abundance of collagen in the samples, which can interfere with the detection of lower-abundance constituents that may be important regulators of cell function. To address this limitation, we developed a novel multi-enzyme digestion approach using treatment with a highly-purified collagenase derived from Clostridium Histolyticum to selectively deplete collagen from ECM-derived protein extracts, reducing its relative abundance from up to 90% to below 10%. Moreover, we applied this new method to characterize the proteome of human decellularized adipose tissue (DAT), human decellularized cancellous bone (DCB), and commercially-available bovine tendon collagen (BTC). We successfully demonstrated with all three sources that collagenase treatment increased the depth of detection and enabled the identification of a variety of signaling proteins that were masked by collagen in standard digestion protocols with trypsin/LysC, increasing the number of proteins identified in the DAT by ∼2.2 fold, the DCB by ∼1.3 fold, and the BTC by ∼1.6 fold. In addition, quantitative proteomics using label-free quantification demonstrated that the DAT and DCB extracts were compositionally distinct, and identified a number of adipogenic and osteogenic proteins that were consistently more highly expressed in the DAT and DCB respectively. Overall, we have developed a new processing method that may be applied in advanced mass spectrometry studies to improve the high-throughput proteomic characterization of bioscaffolds derived from mammalian tissues. Further, our study provides new insight into the complex ECM composition of two human decellularized tissues of interest as cell-instructive platforms for regenerative medicine.

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

Effect of decellularized adipose tissue particle size and cell density on adipose-derived stem cell proliferation and adipogenic differentiation in composite methacrylated chondroitin sulphate hydrogels.

An injectable composite scaffold incorporating decellularized adipose tissue (DAT) as a bioactive matrix within a hydrogel phase capable of in situ polymerization would be advantageous for adipose-derived stem cell (ASC) delivery in the filling of small or irregular soft tissue defects. Building on previous work, the current study investigates DAT milling methods and the effects of DAT particle size and cell seeding density on the response of human ASCs encapsulated in photo-cross-linkable methacrylated chondroitin sulphate (MCS)-DAT composite hydrogels. DAT particles were generated by milling lyophilized DAT and the particle size was controlled through the processing conditions with the goal of developing composite scaffolds with a tissue-specific 3D microenvironment tuned to enhance adipogenesis. ASC proliferation and adipogenic differentiation were assessed in vitro in scaffolds incorporating small (average diameter of 38 ± 6 µm) or large (average diameter of 278 ± 3 µm) DAT particles in comparison to MCS controls over a period of up to 21 d. Adipogenic differentiation was enhanced in the composites incorporating the smaller DAT particles and seeded at the higher density of 5 × 10(5) ASCs/scaffold, as measured by glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, semi-quantitative analysis of perilipin expression and oil red O staining of intracellular lipid accumulation. Overall, this study demonstrates that decellularized tissue particle size can impact stem cell differentiation through cell-cell and cell-matrix interactions, providing relevant insight towards the rational design of composite biomaterial scaffolds for adipose tissue engineering.

Stimulus timing by people with Parkinson's disease.

Previous literature suggests that Parkinson's disease is marked by deficits in timed behaviour. However, the majority of studies of central timing mechanisms in patients with Parkinson's disease have used timing tasks with a motor component. Since the motor abnormalities are a defining feature of the condition, the status of timing in Parkinson's disease remains uncertain. Data are reported from patients with mild to moderate Parkinson's disease (both on and off medication) and age- and IQ-matched controls on a range of stimulus timing tasks without counting. Tasks used were temporal generalization, bisection, threshold determination, verbal estimation, and a memory for duration task. Performance of patients was generally "normal" on all tasks, but significant differences from performance of controls were found on the memory for duration task. Among the "normal" effects noted were arithmetic mean bisection, asymmetric temporal generalization gradients, and subjective shortening on the memory for duration task. The results suggest (a) that some previous reports of timing "deficits" in Parkinson's patients were possibly due to the use of tasks requiring a timed manual response and (b) small differences between patients and controls may be found on tasks where two stimuli are presented on each trial, whether patients are on medication or off it.