Spatially Precise Genetic Engineering at the Electrode-Tissue Interface.

The interface between electrodes and neural tissues plays a pivotal role in determining the efficacy and fidelity of neural activity recording and modulation. While considerable efforts have been made to improve the electrode-tissue interface, the majority of studies have primarily concentrated on the development of biocompatible neural electrodes through abiotic materials and structural engineering. In this study, an approach is presented that seamlessly integrates abiotic and biotic engineering principles into the electrode-tissue interface. Specifically, ultraflexible neural electrodes with short hairpin RNAs (shRNAs) designed to silence the expression of endogenous genes within neural tissues are combined. The system facilitates shRNA-mediated knockdown of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and polypyrimidine tract-binding protein 1 (PTBP1), two essential genes associated in neural survival/growth and neurogenesis, within specific cell populations located at the electrode-tissue interface. Additionally, it is demonstrated that the downregulation of PTEN in neurons can result in an enlargement of neuronal cell bodies at the electrode-tissue interface. Furthermore, the system enables long-term monitoring of neuronal activities following PTEN knockdown in a mouse model of Parkinson's disease and traumatic brain injury. The system provides a versatile approach for genetically engineering the electrode-tissue interface with unparalleled precision, paving the way for the development of regenerative electronics and next-generation brain-machine interfaces.

Free-Standing Carbon Nanotube Embroidered Graphene Film Electrode Array for Stable Neural Interfacing.

Implantable neural probes that are mechanically flexible yet robust are attractive candidates for achieving stable neural interfacing in the brain. Current flexible neural probes consist mainly of metal thin-film electrodes integrated on micrometer-thick polymer substrates, making it challenging to achieve electrode-tissue interfacing on the cellular scale. Here, we describe implantable neural probes that consist of robust carbon nanotube network embroidered graphene (CeG) films as free-standing recording microelectrodes. Our CeG film microelectrode arrays (CeG_MEAs) are ultraflexible yet mechanically robust, thus enabling cellular-scale electrode-tissue interfacing. Chronically implanted CeG_MEAs can stably track the activities of the same population of neurons over two months. Our results highlight the potential of ultraflexible and free-standing carbon nanofilms for stable neural interfacing in the brain.

Ultraflexible Neural Probes for Multidirectional Neuronal Activity Recordings over Large Spatial and Temporal Scales.

The widespread dissemination of ultraflexible neural probes depends on the development of advanced materials and implementation strategies that can allow reliable implantation of ultraflexible neural probes into targeted brain regions, especially deep and difficult-to-access brain regions. Here, we report ultraflexible and multidirectional probes that are encapsulated in a biocompatible polymer alloy with controllable dissolution kinetics. Our probes can be reliably implanted into targeted brain regions over large spatial scales, including deep hindbrain regions that are anatomically difficult-to-access in vivo. Chronically implanted probes can enable long-term, multidirectional recordings from several hundreds of neurons across distributed brain regions. In particular, our results show that 87.0% of chronically recorded neurons in the hindbrain are interneurons, whereas only 41.9% of chronically recorded neurons in the cortex are interneurons. These results demonstrate that our ultraflexible neural probes are a promising tool for large-scale, long-term neural circuit dissection in the brain.

Polymer nanofiber network reinforced gold electrode array for neural activity recording.

Flexible and stretchable neural electrodes are promising tools for high-fidelity interfacing with soft and curvilinear brain surface. Here, we describe a flexible and stretchable neural electrode array that consists of polyacrylonitrile (PAN) nanofiber network reinforced gold (Au) film electrodes. Under stretching, the interweaving PAN nanofibers effectively terminate the formation of propagating cracks in the Au films and thus enable the formation of a dynamically stable electrode-tissue interface. Moreover, the PAN nanofibers increase the surface roughness and active surface areas of the Au electrodes, leading to reduced electrochemical impedance and improved signal-to-noise ratio. As a result, PAN nanofiber network reinforced Au electrode arrays can allow for reliable in vivo multichannel recording of epileptiform activities in rats. Supplementary Information: The online version contains supplementary material available at 10.1007/s13534-022-00257-5.

Self-assembled ultraflexible probes for long-term neural recordings and neuromodulation.

Ultraflexible microelectrode arrays (MEAs) that can stably record from a large number of neurons after their chronic implantation offer opportunities for understanding neural circuit mechanisms and developing next-generation brain-computer interfaces. The implementation of ultraflexible MEAs requires their reliable implantation into deep brain tissues in a minimally invasive manner, as well as their precise integration with optogenetic tools to enable the simultaneous recording of neural activity and neuromodulation. Here, we describe the process for the preparation of elastocapillary self-assembled ultraflexible MEAs, their use in combination with adeno-associated virus vectors carrying opsin genes and promoters to form an optrode probe and their in vivo experimental use in the brains of rodents, enabling electrophysiological recordings and optical modulation of neuronal activity over long periods of time (on the order of weeks to months). The procedures, including device fabrication, probe assembly and implantation, can be completed within 3 weeks. The protocol is intended to facilitate the applications of ultraflexible MEAs for long-term neuronal activity recording and combined electrophysiology and optogenetics. The protocol requires users with expertise in clean room facilities for the fabrication of ultraflexible MEAs.

Electrodeposited NaYF4:Yb3+, Er3+ up-conversion films for flexible neural device construction and near-infrared optogenetics.

Near-infrared optogenetics based on up-conversion materials provides a promising tool for the dissection of neural circuit functions in deep brain regions. However, it remains a challenge to combine near-infrared up-conversion optogenetic stimulation with high-density electrophysiological recording in a minimally invasive manner. Here, we develop a flexible device for simultaneous electrophysiological recording and near-infrared optogenetics. The flexible device is constructed by integrating polymer-based flexible recording microelectrodes with electrodeposited NaYF4:Yb3+, Er3+ up-conversion films that can convert deep-tissue-penetrating near-infrared light into visible light for optogenetic activation of C1V1-expressing neurons. The emission properties of the up-conversion films are optimized for green light emission to stimulate C1V1 opsins. Owing to their minimized surgical footprint and high mechanical compliance, chronically implanted devices enable simultaneous electrophysiological recording and near-infrared optogenetic modulation of neuronal activities in the brain.

Recent developments in multifunctional neural probes for simultaneous neural recording and modulation.

Neural probes are among the most widely applied tools for studying neural circuit functions and treating neurological disorders. Given the complexity of the nervous system, it is highly desirable to monitor and modulate neural activities simultaneously at the cellular scale. In this review, we provide an overview of recent developments in multifunctional neural probes that allow simultaneous neural activity recording and modulation through different modalities, including chemical, electrical, and optical stimulation. We will focus on the material and structural design of multifunctional neural probes and their interfaces with neural tissues. Finally, future challenges and prospects of multifunctional neural probes will be discussed.

PEG-mediated transduction of rAAV as a platform for spatially confined and efficient gene delivery.

BACKGROUND: Recombinant adeno-associated viruses (rAAV) are commonly used vectors for gene delivery in both basic neuroscience and clinical applications due to their nonpathogenic, minimally immunogenic, and sustained expression properties. However, several challenges remain for the wide-scale rAAV applications, including poor infection of many clinically important cell lines, insufficient expression at low titers, and diffusive transduction in vivo. METHODS: In this work, PEG, which is a safe and non-toxic polymer of ethylene oxide monomer, was applied as an auxiliary transduction agent to improve the expression of rAAV. In detail, a small dose of PEG was added into the rAAV solution for the transgene expression in cell lines in vitro, and in the central nervous system (CNS) in vivo. The biocompatibility of PEG enhancer was assessed by characterizing the immune responses, cell morphology, cell tropism of rAAV, neuronal apoptosis, as well as motor function of animals. RESULTS: The results show that small dose of PEG additive can effectively improve the gene expression characteristics of rAAV both in vitro and in vivo. Specifically, the PEG additive allows efficient transgene expression in cell lines that are difficult to be transfected with rAAV alone. In vivo studies show that the PEG additive can promote a spatially confined and efficient transgene expression of low-titer rAAV in the brain over long terms. In addition, no obvious side effects of PEG were observed on CNS in the biocompatibility studies. CONCLUSIONS: This spatially confined and efficient transduction method can facilitate the applications of rAAV in fundamental research, especially in the precise dissection of neural circuits, and also improve the capabilities of rAAV in the treatment of neurological diseases which originate from the disorders of small nuclei in the brain.

A Prognostic Nomogram with High Accuracy Based on 2D-SWE in Patients with Acute-on-chronic Liver Failure.

Background and Aims: Acute-on-chronic liver failure (ACLF) is associated with very high mortality. Accurate prediction of prognosis is critical in navigating optimal treatment decisions to improve patient survival. This study was aimed to develop a new nomogram integrating two-dimensional shear wave elastography (2D-SWE) values with other independent prognostic factors to improve the precision of predicting ACLF patient outcomes. Methods: A total of 449 consecutive patients with ACLF were recruited and randomly allocated to a training cohort (n=315) or a test cohort (n=134). 2D-SWE values, conventional ultrasound features, laboratory tests, and other clinical characteristics were included in univariate and multivariate analysis. Factors with prognostic value were then used to construct a novel prognostic nomogram. Receiver operating curves (ROCs) were generated to evaluate and compare the performance of the novel and published models including the Model for End-Stage Liver Disease (MELD), MELD combined with sodium (MELD-Na), and Jin's model. The model was validated in a prospective cohort (n=102). Results: A ACLF prognostic nomogram was developed with independent prognostic factors, including 2D-SWE, age, total bilirubin (TB), neutrophils (Neu), and the international normalized ratio (INR). The area under the ROC curve (AUC) was 0.849 for the new model in the training cohort and 0.861 in the prospective validation cohort, which were significantly greater than those for MELD (0.758), MELD-Na (0.750), and Jin's model (0.777, all p <0.05). Calibration curve analysis revealed good agreement between the predicted and observed probabilities. The new nomogram had superior overall net benefit and clinical utility. Conclusions: We established and validated a 2D-SWE-based noninvasive nomogram to predict the prognosis of ACLF patients that was more accurate than other prognostic models.

US Shear-Wave Elastography Dispersion for Characterization of Chronic Liver Disease.

Background Little is known about the benefits of the use of dispersion slope (DS) as a viscosity-related parameter derived from two-dimensional (2D) shear-wave elastography (SWE) in the stratification of hepatic pathologic stages. Purpose To evaluate whether DS as an additional parameter can improve the diagnostic performance in detecting liver necroinflammation, fibrosis, and steatosis. Materials and Methods In this prospective study, consecutive participants with chronic liver disease who underwent liver biopsy and 2D SWE were recruited between July 2019 and September 2020. DS and liver stiffness (LS) measurements were obtained with use of a 2D SWE system immediately before biopsy. The biopsy specimens were assessed to obtain the scores of fibrosis, necroinflammation, and steatosis. Differences in the area under the receiver operating characteristic curve (AUC) were used to compare the diagnostic performance of DS, LS, and a combination of DS and LS. Results There were 159 participants evaluated (among them, 79 participants with chronic hepatitis B and 11 participants with nonalcoholic fatty liver disease). The distributions of DS values among various necroinflammatory activities (P = .02) and fibrosis stages (P < .001) were different. Moreover, DS was only associated with fibrosis after subgroup analysis based on the fibrosis stages and necroinflammatory activities (P < .001). The AUCs of DS in detecting clinically significant fibrosis (fibrosis stage ≥F2), cirrhosis (fibrosis stage of F4), and moderate to severe necroinflammatory activity (necroinflammatory activity ≥A2) were 0.72 (95% CI: 0.64, 0.79), 0.71 (95% CI: 0.63, 0.78), and 0.64 (95% CI: 0.55, 0.71), respectively. The differences of AUCs were not apparent for the DS and LS combination model after excluding DS (fibrosis stage ≥F2: 0.00 [95% CI: 0.00, 0.01], fibrosis stage of F4: -0.01 [95% CI: -0.02, 0.00], and necroinflammatory activity ≥A2: 0.00 [95% CI: 0.00, 0.01]). Conclusion The addition of dispersion slope derived from two-dimensional shear-wave elastography did not improve the diagnostic performance in detecting liver fibrosis, necroinflammation, or steatosis in patients with primarily viral hepatitis. ClinicalTrials.gov registration no.: NCT03777293 © RSNA, 2022 Online supplemental material is available for this article.

Comparison between spleen and liver stiffness measurements by sound touch elastography for diagnosing cirrhosis at different aminotransferase levels: a prospective multicenter study.

OBJECTIVES: To compare the performance of spleen stiffness measurement (SSM) and liver stiffness measurement (LSM) by sound touch elastography (STE) for the diagnosis of cirrhosis at different alanine aminotransferase (ALT) levels, and to compare the applicability and repeatability of SSM with LSM performed by STE, a new two-dimensional shear wave elastography technology. METHODS: This prospective multicenter study included 25 centers and recruited chronic hepatitis B (CHB) patients with liver biopsy between May 2018 and November 2019. All patients underwent LSM and SSM by STE. Success and reliability rates were calculated and compared. Intra-observer agreement was assessed using intraclass correlation coefficients (ICCs). Differences between areas under the receiver operating characteristic curves (AUCs) of LSMs and SSMs at different ALT levels were compared using the Delong test. RESULTS: Among 603 CHB patients, the success and reliability rates of SSM were 94.53% (570/603) and 85.74% (517/603), respectively, which were similar to those of LSM (p > 0.05), respectively. The ICC for intra-observer agreements of SSM was 0.964 (p < 0.001). In the total cohort, ALT ≤ 2 × upper limit of normal (ULN) group, and A0-1 group, the AUCs of SSMs were significantly lower than those of LSMs for the diagnosis of cirrhosis (p < 0.001). In the ALT > 2 × ULN group and A2-3 group, the AUC of SSM improved and was not significantly different from that of LSM (p = 0.342, p = 0.510, respectively). CONCLUSIONS: SSM by STE achieved applicability and repeatability equivalent to those of LSM. SSM might be a good substitute to LSM in patients with high ALT levels. KEY POINTS: • Spleen stiffness measurement performed by sound touch elastography was proven to have similar applicability and repeatability to liver stiffness measurement in this prospective multicenter study. • Spleen stiffness measurement demonstrated a poorer diagnostic performance for cirrhosis compared with liver stiffness measurement in the total cohort and low ALT level group, yet it showed a similar diagnostic performance to liver stiffness measurement in patients with high ALT levels.

Usefulness of New Shear Wave Elastography Technique for Noninvasive Assessment of Liver Fibrosis in Patients with Chronic Hepatitis B: A Prospective Multicenter Study.

PURPOSE: To explore the usefulness of liver stiffness measurements (LSMs) by sound touch elastography (STE) and sound touch quantification (STQ) in chronic hepatitis B (CHB) patients for staging fibrosis. METHODS: This prospective multicenter study recruited normal volunteers and CHB patients between May 2018 and October 2019. The volunteers underwent LSM by STE and supersonic shear imaging (SSI) or by STQ and acoustic radiation force impulse imaging (ARFI). CHB patients underwent liver biopsy and LSM by both STE/STQ. The areas under the receiver operating characteristic curves (AUCs) for staging fibrosis were calculated. RESULTS: Overall, 97 volunteers and 524 CHB patients were finally eligible for the study. The successful STE and STQ measurement rates were both 100 % in volunteers and 99.4 % in CHB patients. The intraclass correlation coefficients (ICCs) for the intra-observer stability of STE and STQ (0.94; 0.90) were similar to those of SSI and ARFI (0.95; 0.87), respectively. STE and STQ showed better accuracy than the aspartate aminotransferase-to-platelet ratio index (APRI) and fibrosis-4 index (FIB-4) (AUC: 0.87 vs 0.86 vs 0.73 vs 0.77) in staging cirrhosis. However, both STE and STQ were not superior to APRI and FIB-4 in staging significant fibrosis (AUC: 0.76 vs 0.73 vs 0.70 vs 0.71, all P-values > 0.05). CONCLUSION: STE and STQ are convenient techniques with a reliable LSM value. They have a similar diagnostic performance and are superior to serum biomarkers in staging cirrhosis in CHB patients.

Shear-Wave Dispersion Slope of the Liver: Effect of Study Protocol and Ascites on the Measurement Applicability.

This study aimed to evaluate the shear-wave dispersion (SWD) scanning protocol including the minimum number of measurements and better size of the region of interest (ROI), as well as the influence of ascites on the measurement applicability. Patients who had undergone serial SWD examinations between July 2019 and December 2020 were included. In patients with chronic liver disease (group A), two different ROI sizes were applied, and at least 10 measurements were repeated to determine the minimum number of measurements and better ROI size. In patients with liver failure (group B), failure and unreliable results were compared between patients with and without ascites. A minimum of five measurements when using a 20-mm ROI and six measurements when using a 10-mm ROI were required. Compared with using a 20-mm ROI, a 10-mm ROI showed a higher unreliable rate. The failure and unreliable rates of SWD in patients with ascites were significantly higher than those in patients without ascites. SWD examination required at least five measurements when using a 20-mm ROI and six measurements when using a 10-mm ROI. A larger ROI was associated with higher reliability, and ascites influenced the failure and reliability of the SWD measurement.

Free-Standing Nanofilm Electrode Arrays for Long-Term Stable Neural Interfacings.

Flexible neural electrodes integrated on micrometer-thick polymer substrates offer important opportunities for improving the stability of neuronal activity recordings during cognitive processes. However, the bending stiffness of micrometer-thick polymer substrates is typically two orders of magnitude higher than that of nanofilm electrodes, making it a limiting factor in electrode-tissue interfacings. Here, this limitation is overcome by developing self-assembled nanofilm electrode arrays (NEAs) that consist of high-density, free-standing gold nanofilm electrodes. Chronically implanted NEAs can form intimate and innervated interfaces with neural tissue, enabling stable neuronal activity recordings across multiple brain regions over several months. As an application example, the activities of the same neuronal populations are tracked across odor discrimination reversal learning and it is illustrated how dorsal striatal neurons represent and update stimulus-outcome associations across multiple timescales. The results underscore the potential of free-standing nanoscale materials for interfacing biological systems over long terms.

Blastic plasmacytoid dendritic cell neoplasm with skin and bone marrow involvement: Report of three cases.

BACKGROUND: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematopoietic malignancy. BPDCN is difficult to diagnose because of the overlap in morphologic and immunophenotypic features with various cutaneous lymphatic hematopoietic tumors. CASE SUMMARY: We report on three BPDCN cases, all characterized by skin nodules and examined by histology, immunohistochemical detection, in situ hybridization for Epstein-Barr virus, and follow-up. We also review the relevant literature. All patients were positive for CD56 and negative for Epstein-Barr encoded small RNA. Two patients had bone marrow involvement. Chemotherapy is the main treatment for BPDCN, but case 1 showed bone marrow suppression and case 2 developed recurrence after chemotherapy. Case 1 survived for 7 mo, case 2 for 17 mo, and case 3 for 9 mo. CONCLUSION: An accurate pathological diagnosis is a precondition for treatment, and the diagnosis of BPDCN should be based on a combination of clinical symptoms, pathological characteristics, immunophenotype, and other auxiliary examinations. It is necessary to clarify the clinicopathological features and biological behavior of BPDCN to improve its understanding by both clinicians and pathologists. Case 2 survived significantly longer than the other two cases, suggesting that the treatment received by case 2 was more effective.

Self-assembled multifunctional neural probes for precise integration of optogenetics and electrophysiology.

Optogenetics combined with electrical recording has emerged as a powerful tool for investigating causal relationships between neural circuit activity and function. However, the size of optogenetically manipulated tissue is typically 1-2 orders of magnitude larger than that can be electrically recorded, rendering difficulty for assigning functional roles of recorded neurons. Here we report a viral vector-delivery optrode (VVD-optrode) system for precise integration of optogenetics and electrophysiology in the brain. Our system consists of flexible microelectrode filaments and fiber optics that are simultaneously self-assembled in a nanoliter-scale, viral vector-delivery polymer carrier. The highly localized delivery and neuronal expression of opsin genes at microelectrode-tissue interfaces ensure high spatial congruence between optogenetically manipulated and electrically recorded neuronal populations. We demonstrate that this multifunctional system is capable of optogenetic manipulation and electrical recording of spatially defined neuronal populations for three months, allowing precise and long-term studies of neural circuit functions.

Anti-fouling peptide functionalization of ultraflexible neural probes for long-term neural activity recordings in the brain.

Implantable neural probes constitute an essential tool for neuronal activity recordings in basic neuroscience and also hold great promise for the development of neuroprosthesis to restore lost motor or sensory functions of the body. However, conventional neural probes are susceptible to biofouling because of their physicochemical mismatch with neural tissues, resulting in signal degradation in chronic studies. Here, we describe an ultraflexible neural probe (uFNP) with anti-fouling zwitterionic peptide modification for long-term stable neural activity recordings. The anti-fouling zwitterionic peptide consists of two parts: negatively charged glutamic acid (E) and positively charged lysine (K) formed EKEKEK (EK) head to create a hydration layer that resists protein adsorption on the microelectrodes, and a fragment of laminin formed IKVAV tail to increase the adhesion of the microelectrodes to neuronal cells. We demonstrate that EK-IKVAV modified uFNPs can allow for stable neuronal activity recordings over 16 weeks. Immunohistological studies confirm that EK-IKVAV functionalized uFNPs could induce greatly reduced neuronal cell loss. Collectively, these results suggest that the anti-fouling zwitterionic peptide functionalization of uFNPs provide a promising route to construct biocompatible and stable neural interfaces.

Deep learning radiomics model accurately predicts hepatocellular carcinoma occurrence in chronic hepatitis B patients: a five-year follow-up.

An early and accurate prediction of hepatocellular carcinoma (HCC) is beneficial for individualized treatment and follow-up of chronic hepatitis B (CHB) patients. We aimed to establish a prediction model for HCC by radiomics analysis in CHB patients and compare performance with liver stiffness measurement (LSM) and other clinical prognostic scores. Initially, 1215 patients were included and finally 434 CHB patients with 5-year follow-up were enrolled, 96.3% of them underwent liver biopsy. Deep learning radiomics analysis was performed on 2170 two-dimensional shear wave elastography (2D-SWE) and corresponding B-mode ultrasound (US) images. These high-throughput imaging features were also combined with low-dimensional serological clinical data by deep learning radiomics to establish different HCC prediction models and to overcome challenges of an unbalanced sample. The best model which is simple with high accuracy was selected. Prediction performance of the selected model was compared with LSM and other clinical prognostic scores. During 5-year follow-up, 32 (7.4%) of 434 patients developed HCC. The best prediction model was HCC-R, which included 2D-SWE and B-mode US images, sex and age. This model showed a high predictive value with areas under the receiver operating characteristic curve (AUCs) of 0.981, 0.942 and 0.900 in training, validation and testing cohorts for predicting 5-year prognosis of HCC. These predictive values were significantly higher than that of LSM (AUC: 0.676~0.784, p < 0.05) and better than that of other clinical prognostic scores (AUC: 0.544~0.869). HCC-R radiomics model based on 2D-SWE and B-mode US images, sex and age comprehensively reflected biomechanical and morphological information of patients and can accurately predict HCC occurrence; thus, this model has great value for treatment and follow-up of CHB patients.

Dynamic Contrast-Enhanced Ultrasound Radiomics for Hepatocellular Carcinoma Recurrence Prediction After Thermal Ablation.

PURPOSE: To develop a radiomics model based on dynamic contrast-enhanced ultrasound (CEUS) to predict early and late recurrence in patients with a single HCC lesion ≤ 5 cm in diameter after thermal ablation. PROCEDURES: We enrolled patients who underwent thermal ablation for HCC in our hospital from April 2004 to April 2017. Radiomics based on two branch convolution recurrent network was utilized to analyze preoperative dynamic CEUS image of HCC lesions to establish CEUS model, in comparison to the conventional ultrasound (US), clinical, and combined models. Clinical follow-up of HCC recurrence after ablation were taken as reference standard to evaluate the predicted performance of CEUS model and other models. RESULTS: We finally analyzed 318 patients (training cohort: test cohort = 255:63). The combined model showed better performance for early recurrence than CUES (in training cohort, AUC, 0.89 vs. 0.84, P < 0.001; in test cohort, AUC, 0.84 vs. 0.83, P = 0.272), US (P < 0.001), or clinical model (P < 0.001). For late recurrence prediction, the combined model showed the best performance than the CEUS (C-index, in training cohort, 0.77 vs. 0.76, P = 0.009; in test cohort, 0.77 vs. 0.68, P < 0.001), US (P < 0.001), or clinical model (P < 0.001). CONCLUSIONS: The CEUS model based on dynamic CEUS radiomics performed well in predicting early HCC recurrence after ablation. The combined model combining CEUS, US radiomics, and clinical factors could stratify the high risk of late recurrence.