Immunotherapy reverses glioma-driven dysfunction of immune system homeostasis.

BACKGROUND: Glioma-induced immune dysregulation of the hematopoietic system has been described in a limited number of studies. In this study, our group further demonstrates that gliomas interrupt the cellular differentiation programming and outcomes of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. HSPCs from glioma-bearing mice are reprogrammed and driven towards expansion of myeloid lineage precursors and myeloid-derived suppressor cells (MDSCs) in secondary lymphoid organs. However, we found this expansion is reversed by immunotherapy. Adoptive cellular therapy (ACT) has been demonstrably efficacious in multiple preclinical models of central nervous system (CNS) malignancies, and here we describe how glioma-induced dysfunction is reversed by this immunotherapeutic platform. METHODS: The impact of orthotopic KR158B-luc glioma on HSPCs was evaluated in an unbiased fashion using single cell RNAseq (scRNAseq) of lineage- cells and phenotypically using flow cytometry. Mature myeloid cell frequencies and function were also evaluated using flow cytometry. Finally, ACT containing total body irradiation, tumor RNA-pulsed dendritic cells, tumor-reactive T cells and HSPCs isolated from glioma-bearing or non-tumor-bearing mice were used to evaluate cell fate differentiation and survival. RESULTS: Using scRNAseq, we observed an altered HSPC landscape in glioma-bearing versus non-tumor-bearing mice . In addition, an expansion of myeloid lineage subsets, including granulocyte macrophage precursors (GMPs) and MDSCs, were observed in glioma-bearing mice relative to non-tumor-bearing controls. Furthermore, MDSCs from glioma-bearing mice demonstrated increased suppressive capacity toward tumor-specific T cells as compared with MDSCs from non-tumor-bearing hosts. Interestingly, treatment with ACT overcame these suppressive properties. When HSPCs from glioma-bearing mice were transferred in the context of ACT, we observed significant survival benefit and long-term cures in orthotopic glioma models compared with mice treated with ACT using non-glioma-bearing HSPCs.

Fuel use in small-scale fishing vessels along the southeast coast of India: a comparative study to ascertain possible reasons and potential recommendations for effective management.

Regulating the fuel consumption of small-scale fishing vessels could help to keep global warming well below 1.5 °C and lead to effective management in small-scale fisheries (SSF) of developing countries like India. In this regard, a bottom-up approach was carried out to collect the requisite data to explore the fuel consumption of small-scale fishing vessels along India's southeast coast. Consequently, twenty-four fishing vessels (type A to type X) were grouped into seven categories based on fishing methods. The estimated numerical value of fuel use intensity (FUI) ranging from 0.08 to 0.80 was used to examine the fuel-efficient fishing vessel and engine type. In addition, the estimated revenue on fuel ranging from ₹5625.06/l to ₹218.07/l and annual greenhouse gas (GHG) emissions using the Tier 1 method were used to understand the economic efficiency and GHG emission trend, respectively. The total annual GHG emissions from all the fishing vessels at the selected sites were about 1.25E + 08 t CO2-eq year-1. The result shows that longline-cum-gillnetters, seine-netters, longliners and drift-gillnetters largely contributed to 65% of the annual GHG emissions. By recognizing the factors influencing the fuel consumption of fishing vessels in SSF, this sector could be understood, effectively managed, and performed well. Therefore, the possible reasons were extensively discussed through a comparative approach, and potential recommendations for effective management were made.

Tumor Treating Fields dually activate STING and AIM2 inflammasomes to induce adjuvant immunity in glioblastoma.

Tumor Treating Fields (TTFields), an approved therapy for glioblastoma (GBM) and malignant mesothelioma, employ noninvasive application of low-intensity, intermediate-frequency, alternating electric fields to disrupt the mitotic spindle, leading to chromosome missegregation and apoptosis. Emerging evidence suggests that TTFields may also induce inflammation. However, the mechanism underlying this property and whether it can be harnessed therapeutically are unclear. Here, we report that TTFields induced focal disruption of the nuclear envelope, leading to cytosolic release of large micronuclei clusters that intensely recruited and activated 2 major DNA sensors - cyclic GMP-AMP synthase (cGAS) and absent in melanoma 2 (AIM2) - and their cognate cGAS/stimulator of interferon genes (STING) and AIM2/caspase 1 inflammasomes to produce proinflammatory cytokines, type 1 interferons (T1IFNs), and T1IFN-responsive genes. In syngeneic murine GBM models, TTFields-treated GBM cells induced antitumor memory immunity and a cure rate of 42% to 66% in a STING- and AIM2-dependent manner. Using single-cell and bulk RNA sequencing of peripheral blood mononuclear cells, we detected robust post-TTFields activation of adaptive immunity in patients with GBM via a T1IFN-based trajectory and identified a gene panel signature of TTFields effects on T cell activation and clonal expansion. Collectively, these studies defined a therapeutic strategy using TTFields as cancer immunotherapy in GBM and potentially other solid tumors.

Factors influencing lower extremity amputation outcomes in people with active foot ulceration in regional Australia: A retrospective cohort study.

Australia has the second highest rate of non-traumatic lower extremity amputation (LEA) globally. Australia's large geographical size is one of the biggest challenges facing limb preservation services and may be contributing to LEA. The aim of this study was to determine what factors contribute to the likelihood of LEA in people with active foot ulceration in regional Australia. This retrospective cohort study audited patients with active foot ulceration in a multidisciplinary high risk foot service (HRFS) in regional Australia. Neurological, vascular and wound characteristics were systematically extracted, along with demographic information. Participants were followed for at least 12 months until healing or LEA occurred. Correlations between LEA and clinical and demographic characteristics were assessed using the Pearson's product moment correlation coefficient and chi squared test for independence. Significant variables (p < 0.05) were included in the model. Direct logistic regression assessed the independent contribution of significantly correlated variables on the likelihood of LEA. Of note, 1876 records were hand screened with 476 participants (25%) meeting the inclusion criteria. Geographical distance from the HRFS, toe systolic pressure (TSP), diabetes and infection were all significantly correlated with LEA and included in the logistic regression model. TSP decrease of 1 mmHg (OR 1.02, 95% CI 1.01-1.03), increased geographical distance (1 km) from HRFS (OR 1.006, 95% CI 1.001-1.01) infection (OR 2.08, 95% CI 1.06-4.07) and presence of diabetes (OR 3.77, 95% CI 1.12-12.65) were all significantly associated with increased likelihood of LEA. HRFS should account for the disparity in outcomes between patients living in close proximity to their service, compared to those in rural areas. Optimal management of diabetes, vascular perfusion and control of infection may also contribute to preventing LEA in people with active foot ulceration.

Do toe blood pressures predict healing after minor lower limb amputation in people with diabetes? A systematic review and meta-analysis.

PURPOSE OF STUDY: To investigate toe systolic blood pressure and/or toe-brachial pressure index in predicting healing post minor diabetic foot amputations. KEY METHODS: A systematic search of EMBASE and PubMed (including Medline and The Cochrane Library) was conducted from database inception to 9 March 2020. Two authors independently reviewed and selected relevant studies. Quality was assessed with a modified Critical Appraisal Skill Programme checklist. MAIN RESULTS: Ten studies met the inclusion criteria. Nine studies investigating toe systolic blood pressure reported healing occurred at mean toe systolic blood pressure values ⩾30 mmHg, ranging between 30 and 83.6 mmHg. The meta-analysis (four studies) found toe systolic blood pressure <30 mmHg had 2.09 times the relative risk of non-healing post amputation, compared to toe systolic blood pressure ⩾30 mmHg (relative risk = 2.09, 95% confidence interval: 1.37-3.20, p = 0.001). Two studies investigating toe-brachial pressure index report successful healing where toe-brachial pressure index >0.2, with one study reporting a higher value of 0.8. MAIN CONCLUSIONS: Successful post-amputation healing outcomes were reported at mean toe systolic blood pressure ⩾30 mmHg, and the results varied considerably between the studies. Further research should identify whether variables, including amputation level, method of wound closure and length of post-operative follow-up periods, affect the values of toe systolic blood pressure and toe-brachial pressure index observed in this review.

Obesity and STING1 genotype associate with 23-valent pneumococcal vaccination efficacy.

BACKGROUNDObesity has been associated with attenuated vaccine responses and an increased risk of contracting pneumococcal pneumonia, but no study to our knowledge has assessed the impact of obesity and genetics on 23-valent pneumococcal vaccine (PPSV23) efficacy. We assessed the relationship of obesity (primary analysis) and stimulator of interferon genes (STING1) genotype (secondary analysis) on PPSV23 efficacy.METHODSNonobese (BMI 22-25 kg/m2) and obese participants (BMI ≥30 kg/m2) were given a single dose of PPSV23. Blood was drawn immediately prior to and 4-6 weeks after vaccination. Serum samples were used to assess PPSV23-specific antibodies. STING1 genotypes were identified using PCR on DNA extracted from peripheral blood samples.RESULTSForty-six participants were categorized as nonobese (n = 23; 56.5% women; mean BMI 23.3 kg/m2) or obese (n = 23; 65.2% women; mean BMI 36.3 kg/m2). Obese participants had an elevated fold change in vaccine-specific responses compared with nonobese participants (P < 0.0001). The WT STING1 group (R232/R232) had a significantly higher PPSV23 response than individuals with a single copy of HAQ-STING1 regardless of BMI (P = 0.0025). When WT was assessed alone, obese participants had a higher fold serotype-specific response compared with nonobese participants (P < 0.0001), but no difference was observed between obese and nonobese individuals with 1 HAQ allele (P = 0.693).CONCLUSIONSThese observations demonstrate a positive association between obesity and PPSV23 efficacy specifically in participants with the WT STING1 genotype.TRIAL REGISTRATIONClinicalTrials.gov NCT02471014.FUNDINGThis research was supported by the NIH and the University of Florida MD-PhD Training Program.

Training methods that improve MD-PhD student self-efficacy for clinical research skills.

INTRODUCTION: MD-PhD training programs train physician-scientists to pursue careers involving both clinical care and research, but decreasing numbers of physician-scientists stay engaged in clinical research. We sought to identify current clinical research training methods utilized by MD-PhD programs and to assess how effective they are in promoting self-efficacy for clinical research. METHODS: The US MD-PhD students were surveyed in April-May 2018. Students identified the clinical research training methods they participated in, and self-efficacy in clinical research was determined using a modified 12-item Clinical Research Appraisal Inventory. RESULTS: Responses were received from 61 of 108 MD-PhD institutions. Responses were obtained from 647 MD-PhD students in all years of training. The primary methods of clinical research training included no clinical research training, and various combinations of didactics, mentored clinical research, and a clinical research practicum. Students with didactics plus mentored clinical research had similar self-efficacy as those with didactics plus clinical research practicum. Training activities that differentiated students who did and did not have the clinical research practicum experience and were associated with higher self-efficacy included exposure to Institutional Review Boards and participation in human subject recruitment. CONCLUSIONS: A clinical research practicum was found to be an effective option for MD-PhD students conducting basic science research to gain experience in clinical research skills. Clinical research self-efficacy was correlated with the amount of clinical research training and specific clinical research tasks, which may inform curriculum development for a variety of clinical and translational research training programs, for example, MD-PhD, TL1, and KL2.

Dendritic Cell-Activating Magnetic Nanoparticles Enable Early Prediction of Antitumor Response with Magnetic Resonance Imaging.

Cancer vaccines initiate antitumor responses in a subset of patients, but the lack of clinically meaningful biomarkers to predict treatment response limits their development. Here, we design multifunctional RNA-loaded magnetic liposomes to initiate potent antitumor immunity and function as an early biomarker of treatment response. These particles activate dendritic cells (DCs) more effectively than electroporation, leading to superior inhibition of tumor growth in treatment models. Inclusion of iron oxide enhances DC transfection and enables tracking of DC migration with magnetic resonance imaging (MRI). We show that T2*-weighted MRI intensity in lymph nodes is a strong correlation of DC trafficking and is an early predictor of antitumor response. In preclinical tumor models, MRI-predicted "responders" identified 2 days after vaccination had significantly smaller tumors 2-5 weeks after treatment and lived 73% longer than MRI-predicted "nonresponders". These studies therefore provide a simple, scalable nanoparticle formulation to generate robust antitumor immune responses and predict individual treatment outcome with MRI.

Quantitative characterization of 3D bioprinted structural elements under cell generated forces.

With improving biofabrication technology, 3D bioprinted constructs increasingly resemble real tissues. However, the fundamental principles describing how cell-generated forces within these constructs drive deformations, mechanical instabilities, and structural failures have not been established, even for basic biofabricated building blocks. Here we investigate mechanical behaviours of 3D printed microbeams made from living cells and extracellular matrix, bioprinting these simple structural elements into a 3D culture medium made from packed microgels, creating a mechanically controlled environment that allows the beams to evolve under cell-generated forces. By varying the properties of the beams and the surrounding microgel medium, we explore the mechanical behaviours exhibited by these structures. We observe buckling, axial contraction, failure, and total static stability, and we develop mechanical models of cell-ECM microbeam mechanics. We envision these models and their generalizations to other fundamental 3D shapes to facilitate the predictable design of biofabricated structures using simple building blocks in the future.

How sensitive and specific is continuous-wave Doppler for detecting peripheral arterial disease in people with and without diabetes? A cross-sectional study.

BACKGROUND: Continuous-wave Doppler is frequently used for detecting peripheral arterial disease in patients with diabetes; however, there is limited evidence investigating diagnostic accuracy. This study aimed to determine sensitivity and specificity of continuous-wave Doppler for detecting peripheral arterial disease in populations with, and without, diabetes and to investigate the influence of disease severity on sensitivity of continuous-wave Doppler for detecting peripheral arterial disease. RESULTS: Data from 396 participants were included. Using colour Duplex ultrasound as reference standard (N=66), printed continuous-wave Doppler waveform analysis sensitivity was 81.75% (95% confidence interval: 76.75 to 85.88) and specificity 89.34% (95% confidence interval: 82.62 to 93.67). Printed continuous-wave Doppler waveform analysis sensitivity for peripheral arterial disease was comparable to sensitivity calculated using angiography as the reference standard (81.67%; 95% confidence interval: 69.56 to 90.48). Sensitivity and specificity were unaffected by diabetes diagnosis (n = 176), sensitivity 82.76% (95% confidence interval: 74.86 to 88.55), and specificity 88.33% (95% confidence interval: 77.82 to 94.23). CONCLUSION: Continuous-wave Doppler is a fair assessment tool for peripheral arterial disease in a community-based sample with suspected peripheral arterial disease. Diagnostic accuracy of continuous-wave Doppler for peripheral arterial disease is unaffected by the presence of diabetes.

Diagnostic accuracy of the postexercise ankle-brachial index for detecting peripheral artery disease in suspected claudicants with and without diabetes.

The postexercise ankle-brachial index (ABI) is recommended in patients with normal resting ABI when peripheral artery disease (PAD) is suspected. The aims of this study were to determine the comparative diagnostic accuracy of the resting and postexercise ABI for detecting PAD, and, the effect of the presence of diabetes on these. Three methods of interpretation currently in use were also investigated: a reduction in postexercise ABI by >20% compared to resting ABI, an ABI value of ≤0.90 postexercise, or a reduction in systolic ankle pressure of >30 mmHg postexercise. This retrospective study used colour duplex ultrasound (CDU) as the reference standard. In 278 limbs (whole group), the resting ABI had an overall area under the curve (AUC) of 0.71, with the postexercise ABI yielding a similar diagnostic accuracy of AUC 0.72. In the non-diabetes group ( n=171), the resting ABI had an overall AUC of 0.74 and the postexercise ABI had a similar AUC of 0.76. In the diabetes group ( n=107), overall accuracy was reduced compared to the non-diabetes group, with the resting ABI having an overall AUC of 0.65 and the postexercise ABI yielding a similar accuracy with an AUC of 0.64. The overall diagnostic accuracy of the postexercise ABI for diagnosing PAD was not greatly improved compared to resting ABI. Given the lower overall diagnostic accuracy in the diabetes group, both the resting and the postexercise ABI results in diabetes populations should be interpreted with caution. There is a risk of undiagnosed disease if relying on these results alone to determine lower limb vascular status.

Diagnostic accuracy of resting systolic toe pressure for diagnosis of peripheral arterial disease in people with and without diabetes: a cross-sectional retrospective case-control study.

BACKGROUND: The resting systolic toe pressure (TP) is a measure of small arterial function in the periphery. TP is used in addition to the ankle-brachial index when screening for peripheral arterial disease (PAD) of the lower limb in those with diabetes, particularly in the presence of lower limb medial arterial calcification. It may be used as an adjunct assessment of lower limb vascular function and as a predictor of wound healing. The aim of this study was to determine the diagnostic accuracy of TP for detecting PAD in people with and without diabetes. METHODS: This was a retrospective case-control study. Two researchers extracted information from consecutive patient records, including TP measurements, colour Duplex ultrasound results, demographic information, and medical history. Measures of diagnostic accuracy were determined by receiver operating curve (ROC) analysis, and calculation of sensitivity, specificity, and positive and negative likelihood ratios. RESULTS: Three hundred and nintey-four participants with suspected PAD were included. In the diabetes group (n = 176), ROC analysis of TP for detecting PAD was 0.78 (95%CI: 0.69 to 0.84). In the control group (n = 218), the ROC of TP was 0.73 (95%CI: 0.70 to 0.80). TP had highest sensitivity when anatomical distribution of disease was both proximal and distal (diabetes group: 79.49%, the control group: 82.61%). TP yielded highest sensitivity in mild disease (50-75% stenosis) in diabetes group, (81.82%) and moderate disease (>75% stenosis) in control group (80.77%). CONCLUSIONS: Our findings indicate that TPs are useful to assist in diagnosing PAD in clinical practice, however, results should be interpreted with caution due to the small probability of PAD being present with a negative test.

Helios Controls a Limited Subset of Regulatory T Cell Functions.

A subpopulation (60-70%) of Foxp3(+) regulatory T cells (Tregs) in both mouse and man expresses the transcription factor Helios, but its role in Treg function is still unknown. We generated Treg-specific Helios-deficient mice to examine the function of Helios in Tregs. We show that the selective deletion of Helios in Tregs leads to slow, progressive systemic immune activation, hypergammaglobulinemia, and enhanced germinal center formation in the absence of organ-specific autoimmunity. Helios-deficient Treg suppressor function was normal in vitro, as well as in an in vivo inflammatory bowel disease model. However, Helios-deficient Tregs failed to control the expansion of pathogenic T cells derived from scurfy mice, failed to mediate T follicular regulatory cell function, and failed to control both T follicular helper cell and Th1 effector cell responses. In competitive settings, Helios-deficient Tregs, particularly effector Tregs, were at a disadvantage, indicating that Helios regulates effector Treg fitness. Thus, we demonstrate that Helios controls certain aspects of Treg-suppressive function, differentiation, and survival.

Predators and patterns of within-host growth can mediate both among-host competition and evolution of transmission potential of parasites.

Parasite prevalence shows tremendous spatiotemporal variation. Theory indicates that this variation might stem from life-history characteristics of parasites and key ecological factors. Here, we illustrate how the interaction of an important predator and the schedule of transmission potential of two parasites can explain parasite abundance. A field survey showed that a noncastrating fungus (Metschnikowia bicuspidata) commonly infected a dominant zooplankton host (Daphnia dentifera), while a castrating bacterial parasite (Pasteuria ramosa) was rare. This result seemed surprising given that the bacterium produces many more infectious propagules (spores) than the fungus upon host death. The fungus's dominance can be explained by the schedule of within-host growth of parasites (i.e., how transmission potential changes over the course of infection) and the release of spores from "sloppy" predators (Chaoborus spp., who consume Daphnia prey whole and then later regurgitate the carapace and parasite spores). In essence, sloppy predators create a niche that the faster-schedule fungus currently occupies. However, a selection experiment showed that the slower-schedule bacterium can evolve into this faster-schedule, predator-mediated niche (but pays a cost in maximal spore yield to do so). Hence, our study shows how parasite life history can interact with predation to strongly influence the ecology, epidemiology, and evolution of infectious disease.

Duplex ultrasonography arteriography as first-line investigation for peripheral vascular disease.

INTRODUCTION: The gold standard for evaluation of the lower extremity arterial tree is catheter angiography. Duplex arterial-occlusive imaging or duplex ultrasonography arteriography, a noninvasive technique, is used as the first-line investigation in patients with peripheral vascular disease at our centre. Based on the results of duplex imaging, patients who require angiographic intervention then proceed with simultaneous catheter arteriography and intervention. This study aimed to compare the results of duplex imaging alone as the first-line investigation against the eventual results of catheter angiography, and to assess the impact of the former on patients' clinical outcomes. METHODS: All cases involving patients who underwent duplex imaging followed by angiographic intervention, from May 2008 to February 2009, were discussed at weekly interdisciplinary meetings. Only patients who underwent lower limb imaging were included in the study. Those who were involved in grafts and stent surveillance studies, as well as those with incomplete duplex images were excluded. RESULTS: During the study period, 113 duplex imaging studies of the lower limb followed by percutaneous transluminal angioplasty were performed at our hospital for peripheral vascular disease. The iliac artery was visualised in 40 images, but could not be visualised in 73 images. There was a potential change in management in three cases due to radiological differences between the duplex images and angiography films. CONCLUSION: In our series, duplex imaging was found to be accurate enough to guide initial clinical management of patients with peripheral vascular disease. This modality is the preferred first-line investigation for such patients at our centre.

Disseminated angiosarcoma of the dialysis fistula in 2 patients without kidney transplants.

Angiosarcoma of the dialysis fistula is a rare occurrence. Of the 8 cases of angiosarcoma of the dialysis fistula reported in the literature, all occurred after kidney transplant and long-term immunosuppression therapy. We report 2 cases of disseminated angiosarcoma of the dialysis fistula in hemodialysis patients without concurrent kidney transplants or immunosuppression. Both patients presented with symptoms of pain and bleeding at the site of the thrombosed fistula. Clinicians should be aware that angiosarcoma of the dialysis fistula can occur in patients without kidney transplants.

Primary aortoenteric fistulae: the challenges in diagnosis and review of treatment.

Primary aortoenteric fistula (PAEF) is a potentially fatal condition which poses a considerable diagnostic challenge because of its infrequency and the nonspecific presentation. We report the case of a 61-year old woman who presented to her general practitioner with nonspecific and intermittent rectal bleeding, hematemesis, and weight loss. Four days later, she presented to the emergency department with worsening symptoms. The investigations revealed a new small 3.8-cm abdominal aortic aneurysm on computed axial tomography (CT), however, no evidence of a fistula was observed either on CT scan or endoscopy. Two days later, she became unstable requiring an emergency laparotomy and was taken to the operation theater for an en bloc resection of a segment of the aorta and duodenum with exclusion of the duodenum with an inline reconstruction of the aorta using a Dacron graft for a PAEF. The published data were reviewed to address the issues of optimal diagnostic methods and management of PAEF.

Second world conference on nanomedicine and drug delivery.

The Institute of Holistic Medical Sciences (IHMS, Kottayam, Kerala, India); the Institute of Macromolecular Science and Engineering (IMSE, Kottayam, Kerala, India) and Mathew Ayurveda und Venen Klinik (MUVK, Klegenfurt, Austria) have jointly conducted a 3-day world conference on Nanomedicine and Drug Delivery (WCN 2011) in Kottayam, Kerala, India from 11-13 March 2011. Application of nanotechnology for treatment, diagnosis, monitoring and control of biological systems has been referred to as 'nanomedicine' by the NIH. Research into the rational delivery and targeting of pharmaceutical, therapeutic, and diagnostic agents is at the forefront of projects in nanomedicine. Nanotechnology will also provide devices to examine tissues in minute detail. Biosensors that are smaller than a cell would give us an inside look at cellular function. Tissues could be analyzed down to the molecular level, giving a completely detailed 'snapshot' of cellular, subcellular, and molecular activities. Today, nanotechnology and nanoscience approaches to particle design and formulation are beginning to expand the market for many drugs and are forming the basis for a highly profitable niche within the industry, but some predicted benefits are hyped. This article gives an outlook of the ongoing research projects conducted all over the world, presented at the conference that highlight rational approaches in design and surface engineering of nanoscale vehicles and entities for site-specific drug delivery and medical imaging after parenteral administration. Potential pitfalls or side effects associated with nanoparticles were also discussed.

Arterio-pancreatic syndrome.

Acute pancreatitis is a single-organ disorder that has multi-organ sequelae. As a result, it can have varied presentations. Acute pancreatitis presenting as acute limb ischemia is rare. We present a patient with acute pancreatitis presenting with bilateral lower limb ischemia. The episode of acute pancreatitis resolved but the acute lower limb ischemia precipitated as the pancreatitis progressed, and necessitated bilateral above-knee amputations. We review the literature and discuss the pathogenesis of such a phenomenon.