Longitudinal Muscle Biopsies Reveal Inter- and Intra-Subject Variability in Cancer Cachexia: Paving the Way for Biopsy-Guided Tailored Treatment.

In the rapidly evolving landscape of cancer cachexia research, the development and refinement of diagnostic and predictive biomarkers constitute an ongoing challenge. This study aims to introduce longitudinal muscle biopsies as a potential framework for disease monitoring and treatment. The initial feasibility and safety assessment was performed for healthy mice and rats that received two consecutive muscle biopsies. The assessment was performed by utilizing three different tools. Subsequently, the protocol was also applied in leiomyosarcoma tumor-bearing rats. Longitudinal muscle biopsies proved to be a safe and feasible technique, especially in rat models. The application of this protocol to tumor-bearing rats further affirmed its tolerability and feasibility, while microscopic evaluation of the biopsies demonstrated varying levels of muscle atrophy with or without leukocyte infiltration. In this tumor model, sequential muscle biopsies confirmed the variability of the cancer cachexia evolution among subjects and at different time-points. Despite the abundance of promising cancer cachexia data during the past decade, the full potential of muscle biopsies is not being leveraged. Sequential muscle biopsies throughout the disease course represent a feasible and safe tool that can be utilized to guide precision treatment and monitor the response in cancer cachexia research.

Firing Alterations of Neurons in Alzheimer's Disease: Are They Merely a Consequence of Pathogenesis or a Pivotal Component of Disease Progression?

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder, yet its underlying causes remain elusive. The conventional perspective on disease pathogenesis attributes alterations in neuronal excitability to molecular changes resulting in synaptic dysfunction. Early hyperexcitability is succeeded by a progressive cessation of electrical activity in neurons, with amyloid beta (Aß) oligomers and tau protein hyperphosphorylation identified as the initial events leading to hyperactivity. In addition to these key proteins, voltage-gated sodium and potassium channels play a decisive role in the altered electrical properties of neurons in AD. Impaired synaptic function and reduced neuronal plasticity contribute to a vicious cycle, resulting in a reduction in the number of synapses and synaptic proteins, impacting their transportation inside the neuron. An understanding of these neurophysiological alterations, combined with abnormalities in the morphology of brain cells, emerges as a crucial avenue for new treatment investigations. This review aims to delve into the detailed exploration of electrical neuronal alterations observed in different AD models affecting single neurons and neuronal networks.

Enhancement of the biological activity of hydroxytyrosol through its oxidation by laccase from Trametes versicolor.

The laccase-catalyzed oxidation of hydroxytyrosol (HT) towards the formation of its bioactive oligomer derivatives was investigated. The biocatalytic oligomerization was catalyzed by laccase from Trametes versicolor in aqueous or various water-miscible organic solvents and deep eutectic solvent (DES)-based media. Mass Spectroscopy and Nuclear Magnetic Resonance were used for the characterization of the products. The solvent system used significantly affects the degree of HT oligomerization. The use of 50 % v/v methanol favored the production of the HT dimer, while other organic solvents as well as DESs led to the formation of hydroxytyrosol trimer and other oligomers. In vitro studies showed that the HT dimer exhibits 3- to 4-fold enhanced antibacterial activity against Gram-positive and Gram-negative bacteria compared to the parent compound. Moreover, the ability of HT dimer to inhibit the activity of soybean lipoxygenase and Candida rugosa lipase was 1.5-fold higher than HT, while molecular docking supported these results. Furthermore, HT dimer showed reduced cytotoxicity against HEK293 cells and exhibited a strong ability to inhibit ROS formation. The enhanced bioactivity of HT dimer indicates that this compound could be considered for use in cosmetics, skin-care products, and nutraceuticals.

Carpal Tunnel Syndrome Automated Diagnosis: A Motor vs. Sensory Nerve Conduction-Based Approach.

The objective of this study was to evaluate the effectiveness of machine learning classification techniques applied to nerve conduction studies (NCS) of motor and sensory signals for the automatic diagnosis of carpal tunnel syndrome (CTS). Two methodologies were tested. In the first methodology, motor signals recorded from the patients' median nerve were transformed into time-frequency spectrograms using the short-time Fourier transform (STFT). These spectrograms were then used as input to a deep two-dimensional convolutional neural network (CONV2D) for classification into two categories: patients and controls. In the second methodology, sensory signals from the patients' median and ulnar nerves were subjected to multilevel wavelet decomposition (MWD), and statistical and non-statistical features were extracted from the decomposed signals. These features were utilized to train and test classifiers. The classification target was set to three categories: normal subjects (controls), patients with mild CTS, and patients with moderate to severe CTS based on conventional electrodiagnosis results. The results of the classification analysis demonstrated that both methodologies surpassed previous attempts at automatic CTS diagnosis. The classification models utilizing the motor signals transformed into time-frequency spectrograms exhibited excellent performance, with average accuracy of 94%. Similarly, the classifiers based on the sensory signals and the extracted features from multilevel wavelet decomposition showed significant accuracy in distinguishing between controls, patients with mild CTS, and patients with moderate to severe CTS, with accuracy of 97.1%. The findings highlight the efficacy of incorporating machine learning algorithms into the diagnostic processes of NCS, providing a valuable tool for clinicians in the diagnosis and management of neuropathies such as CTS.

Cell replacement therapy with stem cells in multiple sclerosis, a systematic review.

Multiple sclerosis (MS) is a chronic inflammatory, autoimmune, and neurodegenerative disease of the central nervous system (CNS), characterized by demyelination and axonal loss. It is induced by attack of autoreactive lymphocytes on the myelin sheath and endogenous remyelination failure, eventually leading to accumulation of neurological disability. Disease-modifying agents can successfully address inflammatory relapses, but have low efficacy in progressive forms of MS, and cannot stop the progressive neurodegenerative process. Thus, the stem cell replacement therapy approach, which aims to overcome CNS cell loss and remyelination failure, is considered a promising alternative treatment. Although the mechanisms behind the beneficial effects of stem cell transplantation are not yet fully understood, neurotrophic support, immunomodulation, and cell replacement appear to play an important role, leading to a multifaceted fight against the pathology of the disease. The present systematic review is focusing on the efficacy of stem cells to migrate at the lesion sites of the CNS and develop functional oligodendrocytes remyelinating axons. While most studies confirm the improvement of neurological deficits after the administration of different stem cell types, many critical issues need to be clarified before they can be efficiently introduced into clinical practice.

Hydroxytyrosol produced by engineered Escherichia coli strains activates Nrf2/HO-1 pathway: An in vitro and in vivo study.

This study explores the biological effects of hydroxytyrosol (HT), produced by the metabolic engineering of Escherichia coli, in a series of in vitro and in vivo experiments. In particular, a metabolically engineered Escherichia coli strain capable of producing HT was constructed and utilized. HEK293 and HeLa cells were exposed to purified HT to determine non-toxic doses that can offer protection against oxidative stress (activation of Nrf2/HO-1 signaling pathway). Male CD-1 mice were orally supplemented with HT to evaluate (1) renal and hepatic toxicity, (2) endogenous system antioxidant response, and (3) activation of Nrf2/HO-1 system in the liver. HT protected cells from oxidative stress through the activation of Nrf2 regulatory network. Activation of Nrf2 signaling pathway was also observed in the hepatic tissue of the mice. HT supplementation was safe and produced differential effects on mice's endogenous antioxidant defense system. HT biosynthesized from genetically modified Escherichia coli strains is an alternative method to produce high-quality HT that exerts favorable effects in the regulation of the organism's response to oxidative stress. Nonetheless, further investigation of the multifactorial action of HT on the antioxidant network regulation is needed.

Cellular Localization of Orexin 1 Receptor in Human Hypothalamus and Morphological Analysis of Neurons Expressing the Receptor.

The orexin system is related to food behavior, energy balance, wakefulness and the reward system. It consists of the neuropeptides orexin A and B, and their receptors, orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R). OX1R has selective affinity for orexin A, and is implicated in multiple functions, such as reward, emotions, and autonomic regulation. This study provides information about the OX1R distribution in human hypothalamus. The human hypothalamus, despite its small size, demonstrates a remarkable complexity in terms of cell populations and cellular morphology. Numerous studies have focused on various neurotransmitters and neuropeptides in the hypothalamus, both in animals and humans, however, there is limited experimental data on the morphological characteristics of neurons. The immunohistochemical analysis of the human hypothalamus revealed that OX1R is mainly found in the lateral hypothalamic area, the lateral preoptic nucleus, the supraoptic nucleus, the dorsomedial nucleus, the ventromedial nucleus, and the paraventricular nucleus. The rest of the hypothalamic nuclei do not express the receptor, except for a very low number of neurons in the mammillary bodies. After identifying the nuclei and neuronal groups that were immunopositive for OX1R, a morphological and morphometric analysis of those neurons was conducted using the Golgi method. The analysis revealed that the neurons in the lateral hypothalamic area were uniform in terms of their morphological characteristics, often forming small groups of three to four neurons. A high proportion of neurons in this area (over 80%) expressed the OX1R, with particularly high expression in the lateral tuberal nucleus (over 95% of neurons). These results were analyzed, and shown to represent, at the cellular level, the distribution of OX1R, and we discuss the regulatory role of orexin A in the intra-hypothalamic areas, such as its special role in the plasticity of neurons, as well as in neuronal networks of the human hypothalamus.

Clinical Evaluation in Parkinson's Disease: Is the Golden Standard Shiny Enough?

Parkinson's disease (PD) has become the second most common neurodegenerative condition following Alzheimer's disease (AD), exhibiting high prevalence and incident rates. Current care strategies for PD patients include brief appointments, which are sparsely allocated, at outpatient clinics, where, in the best case scenario, expert neurologists evaluate disease progression using established rating scales and patient-reported questionnaires, which have interpretability issues and are subject to recall bias. In this context, artificial-intelligence-driven telehealth solutions, such as wearable devices, have the potential to improve patient care and support physicians to manage PD more effectively by monitoring patients in their familiar environment in an objective manner. In this study, we evaluate the validity of in-office clinical assessment using the MDS-UPDRS rating scale compared to home monitoring. Elaborating the results for 20 patients with Parkinson's disease, we observed moderate to strong correlations for most symptoms (bradykinesia, rest tremor, gait impairment, and freezing of gait), as well as for fluctuating conditions (dyskinesia and OFF). In addition, we identified for the first time the existence of an index capable of remotely measuring patients' quality of life. In summary, an in-office examination is only partially representative of most PD symptoms and cannot accurately capture daytime fluctuations and patients' quality of life.

Does Green Exfoliation of Graphene Produce More Biocompatible Structures?

Graphene has been studied thoroughly for its use in biomedical applications over the last decades. A crucial factor for a material to be used in such applications is its biocompatibility. Various factors affect the biocompatibility and toxicity of graphene structures, including lateral size, number of layers, surface functionalization, and way of production. In this work, we tested that the green production of few-layer bio-graphene (bG) enhances its biocompatibility compared to chemical-graphene (cG). When tested against three different cell lines in terms of MTT assays, both materials proved to be well-tolerated at a wide range of doses. However, high doses of cG induce long-term toxicity and have a tendency for apoptosis. Neither bG nor cG induced ROS generation or cell cycle modifications. Finally, both materials affect the expression of inflammatory proteins such as Nrf2, NF-kB and HO-1 but further research is required for a safe result. In conclusion, although there is little to choose between bG and cG, bG's sustainable way of production makes it a much more attractive and promising candidate for biomedical applications.

Oral Supplementation with Hydroxytyrosol Synthesized Using Genetically Modified Escherichia coli Strains and Essential Oils Mixture: A Pilot Study on the Safety and Biological Activity.

Several natural compounds have been explored as immune-boosting, antioxidant and anti-inflammatory dietary supplements. Amongst them, hydroxytyrosol, a natural antioxidant found in olive products, and endemic medicinal plants have attracted the scientific community's and industry's interest. We investigated the safety and biological activity of a standardised supplement containing 10 mg of hydroxytyrosol synthesized using genetically modified Escherichia coli strains and equal amounts (8.33 µL) of essential oils from Origanum vulgare subsp. hirtum, Salvia fruticosa and Crithmum maritimum in an open-label, single-arm, prospective clinical study. The supplement was given to 12 healthy subjects, aged 26-52, once a day for 8 weeks. Fasting blood was collected at three-time points (weeks 0, 8 and follow-up at 12) for analysis, which included full blood count and biochemical determination of lipid profile, glucose homeostasis and liver function panel. Specific biomarkers, namely homocysteine, oxLDL, catalase and total glutathione (GSH) were also studied. The supplement induced a significant reduction in glucose, homocysteine and oxLDL levels and was tolerated by the subjects who reported no side effects. Cholesterol, triglyceride levels and liver enzymes remained unaffected except for LDH. These data indicate the supplement's safety and its potential health-beneficial effects against pathologic conditions linked to cardiovascular disease.

Oxidized-Multiwalled Carbon Nanotubes as Non-Toxic Nanocarriers for Hydroxytyrosol Delivery in Cells.

Carbon nanotubes (CNTs) possess excellent physicochemical and structural properties alongside their nano dimensions, constituting a medical platform for the delivery of different therapeutic molecules and drug systems. Hydroxytyrosol (HT) is a molecule with potent antioxidant properties that, however, is rapidly metabolized in the organism. HT immobilized on functionalized CNTs could improve its oral absorption and protect it against rapid degradation and elimination. This study investigated the effects of cellular oxidized multiwall carbon nanotubes (oxMWCNTs) as biocompatible carriers of HT. The oxidation of MWCNTs via H2SO4 and HNO3 has a double effect since it leads to increased hydrophilicity, while the introduced oxygen functionalities can contribute to the delivery of the drug. The in vitro effects of HT, oxMWCNTS, and oxMWCNTS functionalized with HT (oxMWCNTS_HT) were studied against two different cell lines (NIH/3T3 and Tg/Tg). We evaluated the toxicity (MTT and clonogenic assay), cell cycle arrest, and reactive oxygen species (ROS) formation. Both cell lines coped with oxMWCNTs even at high doses. oxMWCNTS_HT acted as pro-oxidants in Tg/Tg cells and as antioxidants in NIH/3T3 cells. These findings suggest that oxMWCNTs could evolve into a promising nanocarrier suitable for targeted drug delivery in the future.

Is graphene the rock upon which new era continuous glucose monitors could be built?

Diabetes mellitus' (DM) prevalence worldwide is estimated to be around 10% and is expected to rise over the next decades. Monitoring blood glucose levels aims to determine whether glucose targets are met to minimize the risk for the development of symptoms related to high or low blood sugar and avoid long-term diabetes complications. Continuous glucose monitoring (CGMs) systems emerged almost two decades ago and have revolutionized the way diabetes is managed. Especially in Type 1 DM, the combination of a CGM with an insulin pump (known as a closed-loop system or artificial pancreas) allows an autonomous regulation of patients' insulin with minimal intervention from the user. However, there is still an unmet need for high accuracy, precision and repeatability of CGMs. Graphene was isolated in 2004 and found immediately fertile ground in various biomedical applications and devices due to its unique combination of properties including its high electrical conductivity. In the last decade, various graphene family nanomaterials have been exploited for the development of enzymatic and non-enzymatic biosensors to determine glucose in biological fluids, such as blood, sweat, and so on. Although great progress has been achieved in the field, several issues need to be addressed for graphene sensors to become a predominant material in the new era of CGMs.

Binuclear VIV/V, MoVI and ZnII - hydroquinonate complexes: Synthesis, stability, oxidative activity and anticancer properties.

Since the discovery of the anticancer properties of cis-platin the road for the development of less toxic and more specific metal ion based anticancer drugs has opened. Based on the low toxicity of VIV/V, MoVI and ZnII metal ions, their binuclear hydroquinonate complexes have been synthesized and their biological activity towards their anticancer properties on various cancerous and non-cancerous cell lines has been evaluated. The new complexes of ZnII with the ligands 2,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)benzene-1,4-diol (H2bpymah) and 2,2'-(((2,5-dihydroxy-1,4-phenylene)bis(methylene))bis((carboxymethyl)ammoniumdiyl))diacetate (H6bicah) have been synthesized and characterized by X-ray crystallography in solid state and 1H NMR in aqueous solution. The binuclear nature of the complexes increases their hydrolytic stability in aqueous solutions at pD 7.0, depending on the metal ion. The most hydrolytic stable VV and ZnII hydroquinonate complexes show to activate O2 towards oxidation of mercaptoethanol in aqueous solutions at physiological pHs. Only the strongest oxidant, the VV complex with bicah6-, significantly activates the intracellular radical oxygen species (ROS) generation. Apparently, the mercaptoethanol oxidation experiment vs time can be used as a preliminary experiment for the prediction of the in vitro ROS generation activity of the complexes in aqueous solutions.

New treatment approaches for Alzheimer's disease: preclinical studies and clinical trials centered on antidiabetic drugs.

INTRODUCTION: Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) represent two major chronic diseases that affect a large percentage of the population and share common pathogenetic mechanisms, including oxidative stress and inflammation. Considering their common mechanistic aspects, and given the current lack of effective therapies for AD, accumulating research has focused on the therapeutic potential of antidiabetic drugs in the treatment or prevention of AD. AREAS COVERED: This review examines the latest preclinical and clinical evidence on the potential of antidiabetic drugs as candidates for AD treatment. Numerous approved drugs for T2DM, including insulin, metformin, glucagon-like peptide-1 receptor agonists (GLP-1 RA), and sodium glucose cotransporter 2 inhibitors (SGLT2i), are in the spotlight and may constitute novel approaches for AD treatment. EXPERT OPINION: Among other pharmacologic agents, GLP-1 RA and SGLT2i have so far exhibited promising results as novel treatment approaches for AD, while current research has centered on deciphering their action on the central nervous system (CNS). Further investigation is crucial to reveal the most effective pharmacological agents and their optimal combinations, maximize their beneficial effects on neurons, and find ways to increase their distribution to the CNS.

Production of hydroxytyrosol rich extract from Olea europaea leaf with enhanced biological activity using immobilized enzyme reactors.

As olive leaves constitute the main by-product of the olive oil industry with important environmental and economic impact, there is an increasing demand for its valorization. In the present work, we report the development and application of immobilized enzyme batch bioreactors for the chemo-enzymatic treatment of an aqueous Olea europaea leaf extract rich in oleuropein to produce an extract enriched in hydroxytyrosol and other oleuropein hydrolysis products. To this end, a robust biocatalyst was developed through the immobilization of ß-glucosidase on chitosan-coated magnetic beads which exhibited high hydrolytic stability after 240 h of incubation at 37 °C. The biocatalyst was successfully used in both a rotating bed-reactor and a stir-tank reactor for the modification of the olive leaf extract leading to high conversion yields of oleuropein (exceeding 90%), while an up to 2.5 times enrichment in hydroxytyrosol was achieved. Over 20 phenolic compounds (from different classes of phytochemicals such as flavonoids, secoiridoids, and their derivatives) were identified, in the extract before and after its modification through various chromatographic and spectroscopic techniques. Finally, the biological activity of both extracts was evaluated. Compared to the non-modified extract, the modified one demonstrated 20% higher antioxidant activity, seven-fold higher antibacterial activity, and enhanced cytotoxicity against leiomyosarcoma cells.

Clinical features and diagnostic tools in idiopathic inflammatory myopathies.

Idiopathic inflammatory myopathies (IIMs) are rare autoimmune disorders affecting primarily muscles, but other organs can be involved. This review describes the clinical features, diagnosis and treatment for IIMs, namely polymyositis (PM), dermatomyositis (DM), sporadic inclusion body myositis (sIBM), immune-mediated necrotizing myopathy (IMNM), and myositis associated with antisynthetase syndrome (ASS). The diagnostic approach has been updated recently based on the discovery of circulating autoantibodies, which has enhanced the management of patients. Currently, validated classification criteria for IIMs allow clinical studies with well-defined sets of patients but diagnostic criteria to guide the care of individual patients in routine clinical practice are still missing. This review analyzes the clinical manifestations and laboratory findings of IIMs, discusses the efficiency of modern and standard methods employed in their workup, and delineates optimal practice for clinical care. Α multidisciplinary diagnostic approach that combines clinical, neurologic and rheumatologic examination, evaluation of electrophysiologic and morphologic muscle characteristics, and assessment of autoantibody immunoassays has been determined to be the preferred approach for effective management of patients with suspected IIMs.

Trends of nanotechnology in type 2 diabetes mellitus treatment.

There are several therapeutic approaches in type 2 diabetes mellitus (T2DM). When diet and exercise fail to control hyperglycemia, patients are forced to start therapy with antidiabetic agents. However, these drugs present several drawbacks that can affect the course of treatment. The major disadvantages of current oral modalities for the treatment of T2DM are mainly depicted in the low bioavailability and the immediate release of the drug, generating the need for an increase in frequency of dosing. In conjugation with the manifestation of adverse side effects, patient compliance to therapy is reduced. Over the past few years nanotechnology has found fertile ground in the development of novel delivery modalities that can potentially enhance anti-diabetic regimes efficacy. All efforts have been targeted towards two main vital steps: (a) to protect the drug by encapsulating it into a nano-carrier system and (b) efficiently release the drug in a gradual as well as controllable manner. However, only a limited number of studies published in the literature used in vivo techniques in order to support findings. Here we discuss the current disadvantages of modern T2DM marketed drugs, and the nanotechnology advances supported by in vivo in mouse/rat models of glucose homeostasis. The generation of drug nanocarriers may increase bioavailability, prolong release and therefore reduce dosing and thus, improve patient compliance. This novel approach might substantially improve quality of life for diabetics. Application of metal nanoformulations as indirect hypoglycemic agents is also discussed.

Effects of secondary biological treatment plant effluent administration, as drinking water, to rats' urogenital system in relation to cadmium and lead accumulation.

The aim of this study was to examine the effect of the secondary biological treatment plant effluent administration on the kidneys, urinary bladder, and testis of Wistar rats in relation to lead (Pb) and cadmium (Cd) accumulation, since such an effluent is used for irrigation of edible plants. Male Wistar rats, randomly assigned into 5 groups, were treated with domestic sewage effluent (DSE) for 24 months. Cadmium and lead concentrations in the DSE, rats' tissues, and urine were estimated by means of atomic spectroscopy. Lead was rapidly accumulated in high amounts in rats' kidney and to a lesser extent in the testis whereas Cd concentration was raised in all tissues examined. Deposition of Cd and Pd in the kidney of the rats resulted in profound damage over time. The results showed that long-term administration to DSE as drinking water exposes living organisms to urogenital stress related to heavy metal concentration and pH of the effluent.

Antitumor effects of the electromagnetic resonant frequencies derived from the 1H NMR spectrum of Ph3Sn(Mercaptonicotinic)SnPh3 complex.

The aim of this article is to investigate the potential cytotoxic and antitumor effects of the resonant electromagnetic fields (rEMFs) derived from the 1H NMR spectrum of the Ph3Sn(Mercaptonicotinic)SnPh3 complex (SnMNA). The ability of the complex's rEMFs to induce leiomyosarcoma (LMS) cell death and to recess tumor (leiomyosarcoma) development in Wistar rats was evaluated. The effects of the simultaneous administration of the SnMNA complex at extremely low concentrations and exposure to its rEMFs was also investigated. The emission of the 1H NMR spectrum of the complex alone or in a combination with low ineffective doses of the complex decreased LMS cell viability mainly through apoptosis. Moreover, the results from the in vivo experiments showed a significant prolongation of life expectancy in tumor-bearing rats exposed to the rEMFs alongside a deceleration in tumor growth rate. We speculate that the rEMFs of a biologically active substance could exert similar biological effects as the substance itself, mainly when is combined with extremely low ineffective concentrations of the substance.

Vanillylmandelic acid protects against reperfusion injury in an experimental animal model of myocardial infarction.

Vanillylmandelic acid, a catecholamine end-metabolite, has been shown to have several biological properties in previous studies, despite considered biologically inactive. We examined the potential effects of vanillylmandelic acid on the ischemic heart following myocardial infarction and reperfusion on a rat model. Thirty-four female Wistar rats were randomized into two groups, control and experimental. They were anesthetized and subjected to myocardial infarction through left anterior descending artery ligation. A previously studied dose of vanillylmandelic acid (10 mg/kg) was administered and the following parameters were studied during ischemia and reperfusion: a) mortality b) severity of ventricular tachyarrhythmias c) premature ventricular contractions and d) heart rate. Administration of vanillymandelic acid significantly reduced the severity of ventricular tachyarrhythmias and mortality rate during reperfusion, while it did not affect any other of the parameters studied. In conclusion, reperfusion injury was blunted through vanillylmandelic acid administration, which seems to be mediated by parasympathetic activation.