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Background: Today, many research groups in the world are struggling to fully understand the mechanisms leading to the carcinogenesis of hazardous mineral fibres, like asbestos, in view of devising effective cancer prevention strategies and therapies. Along this research line, our work attempts the completion of a model aimed at evaluating how, and to what extent, physical-crystal-chemical and morphological parameters of mineral fibres prompt adverse effects in vivo leading to carcinogenesis., Methods: In vitro toxicology tests that deliver information on the 10 key characteristics of carcinogens adopted by the International Association for Research on Cancer (IARC) have been systematically collected for a commercial chrysotile, standard UICC crocidolite and wollastonite. The analysis of the in vitro data allowed us to assess the major fibre parameters responsible for alterations in the key characteristics of carcinogens for each investigated fibre and the intensity of their effect., Results: Crystal habit and density of the fibres affect exposure but are not major parameters contributing to the KCs. For chrysotile, besides length, we found that fibre parameters that greatly contribute to the KCs are the surface area and the dissolution rate with the related velocity of release of metals (namely iron). For crocidolite, they are the fibre length, iron content and related parameters like the ferrous iron content, iron nuclearity, transition metals content and zeta potential., Conclusions: The results of our study can be a starting point for developing personalized cancer screening and prevention strategies as long as the nature of the fibre of the exposed patient is known. We can speculate on a future personalized prevention therapy targeting the fibres with surface-engineered nanocarriers with active complexes that are selective for the surface charge of the fibres. For chrysotile, a complex with deferasirox that can chelate Fe 2+ and deferoxamine that preferentially chelates Fe 3+ is proposed with the anchorage to the silica chrysotile surface driven by aspartic acid. For crocidolite, deferiprone chelating both Fe 3+ and Fe 2+ combined with lysine to attract the silica crocidolite surface is proposed., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Background/objectives: Three-dimensional (3D) cell culture technologies allow us to overcome the constraints of two-dimensional methods in different fields like biochemistry and cell biology and in pharmaceutical in vitro tests. In this study, a novel 3D hydrogel sponge scaffold, composed of a crosslinked polyacrylic acid forming a porous matrix, has been developed and characterized., Methods: The scaffold was obtained via an innovative procedure involving thermal treatment followed by a salt-leaching step on a matrix-containing polymer along with a gas-forming agent. Based on experimental design for mixtures, a series of formulations were prepared to study the effect of the three components (polyacrylic acid, NaHCO 3 and NaCl) on the scaffold mechanical properties, density, swelling behavior and morphological changes. Physical appearance, surface morphology, porosity, molecular diffusion, transparency, biocompatibility and cytocompatibility were also evaluated., Results: The hydrogel scaffolds obtained show high porosity and good optical transparency and mechanical resistance. The scaffolds were successfully employed to culture several cell lines for more than 20 days., Conclusions: The developed scaffolds could be an important tool, as such or with a specific coating, to obtain a more predictive cellular response to evaluate drugs in preclinical studies or for testing chemical compounds, biocides and cosmetics, thus reducing animal testing.

Background/objectives: Oral diseases causing mucosal lesions are normally treated with local or systemic anti-inflammatory, analgesic and antimicrobial agents. The development of topical formulations, including wound-healing promoters, might speed up the recovery process, improving patients' quality of life, and reduce the risk of deterioration in health conditions. In this study, a mucoadhesive multilayer film, including a novel biocompatible substance (solubilized eggshell membrane, SESM), was rationally designed., Methods: The SESM preparation procedure was optimized and its biological effects on cell proliferation and inflammation marker gene expression were evaluated in vitro; preformulation studies were conducted to identify the most promising polymers with film-forming properties; then, trilayer films, consisting of an outer layer including chlorhexidine digluconate as a model drug, a supporting layer and a mucoadhesive layer, incorporating SESM, were prepared using the casting method and their mechanical, adhesion and drug release control properties were evaluated., Results: SESM proved to possess a notable wound-healing capacity, inducing a wound closure of 84% in 24 h without inhibiting blood clotting. The films revealed a maximum detachment force from porcine mucosa of approx. 1.7 kPa and maximum in vivo residence time of approx. 200-240 min; finally, they released up to 98% of the loaded drug within 4 h., Conclusions: The formulated trilayer films were found to possess adequate properties, making them potentially suitable for protecting oral lesions and favoring their rapid healing, while releasing antimicrobial substances that might be beneficial in reducing the risk of bacterial infections.

This work is an in vitro toxicity study of two asbestiform erionites from Kaipara and Gawler Downs in New Zealand. This study is the first, to the knowledge of the authors, to investigate the mechanisms that trigger adverse effects leading to carcinogenicity from New Zealand erionites. The effects induced by the erionite fibres from New Zealand were compared with those produced by positive (crocidolite) and negative (wollastonite) standards, and other erionite fibres described in the literature. The cytotoxicity/genotoxicity/inflammatory potential was determined by: (i) analysis of the cytotoxic potential by MTT tests on human cell lines mimicking primary cells making direct contact with fibres in the lungs, combined with apoptosis tests and cell membrane damage by fluorescence microscopy analyses; (ii) analysis of the genotoxic potential by quantification of DNA damage measuring double strand break foci by γ-H2AX nuclear staining in confocal microscopy analyses; (iii) analyses of the acute (24-72h) and early-chronic (7d) inflammatory effect by gene expression analyses of several cytokines, as well as of fibrotic and Epithelial to Mesenchymal transition (EMT) markers. The intensity of cell responses to these erionites are comparable to that of standard carcinogenic crocidolite, indicating that the two erionite fibres exhibit a significant acute toxic potential, with a particular alarming effect from the Gawler Downs sample from South Island. Our results confirm that the investigated erionites from New Zealand may represent an environmental hazard. However, further investigation is required to determine potential environmental exposure pathways by which erionite may become airborne and assess any environmental risks that may arise., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Martin Brook reports financial support was provided by New Zealand Ministry of Business Innovation and Employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Glaucoma is the second leading cause of irreversible blindness worldwide. Although genetic background contributes differently to rare early-onset glaucoma (before age 40) or common adult-onset glaucoma, it is now considered an important factor in all major forms of the disease. Genetic and genomic studies, including GWAS, are contributing to identifying novel loci associated with glaucoma or to endophenotypes across ancestries to enrich the knowledge about glaucoma genetic susceptibility. Moreover, new high-throughput functional genomics contributes to defining the relevance of genetic results in the biological pathways and processes involved in glaucoma pathogenesis. Such studies are expected to advance significantly our understanding of glaucoma's genetic basis and provide new druggable targets to treat glaucoma. This review gives an overview of the role of genetics in the pathogenesis or risk of glaucoma., Competing Interests: The authors declare that the review was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tirendi, Domenicotti, Bassi and Vernazza.)

Introduction: The latest GLOBOCAN 2021 reports that colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Most CRC cases are sporadic and associated with several risk factors, including lifestyle habits, gut dysbiosis, chronic inflammation, and oxidative stress., Aim: To summarize the biology of CRC and discuss current therapeutic interventions designed to counteract CRC development and to overcome chemoresistance., Methods: Literature searches were conducted using PubMed and focusing the attention on the keywords such as "Current treatment of CRC" or "chemoresistance and CRC" or "oxidative stress and CRC" or "novel drug delivery approaches in cancer" or "immunotherapy in CRC" or "gut microbiota in CRC" or "systematic review and meta-analysis of randomized controlled trials" or "CSCs and CRC". The citations included in the search ranged from September 1988 to December 2022. An additional search was carried out using the clinical trial database., Results: Rounds of adjuvant therapies, including radiotherapy, chemotherapy, and immunotherapy are commonly planned to reduce cancer recurrence after surgery (stage II and stage III CRC patients) and to improve overall survival (stage IV). 5-fluorouracil-based chemotherapy in combination with other cytotoxic drugs, is the mainstay to treat CRC. However, the onset of the inherent or acquired resistance and the presence of chemoresistant cancer stem cells drastically reduce the efficacy. On the other hand, the genetic-molecular heterogeneity of CRC often precludes also the efficacy of new therapeutic approaches such as immunotherapies. Therefore, the CRC complexity made of natural or acquired multidrug resistance has made it necessary the search for new druggable targets and new delivery systems., Conclusion: Further knowledge of the underlying CRC mechanisms and a comprehensive overview of current therapeutic opportunities can provide the basis for identifying pharmacological and biological barriers that render therapies ineffective and for identifying new potential biomarkers and therapeutic targets for advanced and aggressive CRC., Competing Interests: The authors declare that the review was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tirendi, Marengo, Domenicotti, Bassi, Almonti and Vernazza.)

Background: Malignant melanoma is the most lethal form of skin cancer which shows BRAF mutation in 50% of patients. In this context, the identification of BRAF V600E mutation led to the development of specific inhibitors like PLX4032. Nevertheless, although its initial success, its clinical efficacy is reduced after six-months of therapy leading to cancer relapse due to the onset of drug resistance. Therefore, investigating the mechanisms underlying PLX4032 resistance is fundamental to improve therapy efficacy. In this context, several models of PLX4032 resistance have been developed, but the discrepancy between in vitro and in vivo results often limits their clinical translation., Methods: The herein reported model has been realized by treating with PLX4032, for six months, patient-derived BRAF-mutated melanoma cells in order to obtain a reliable model of acquired PLX4032 resistance that could be predictive of patient's treatment responses. Metabolic analyses were performed by evaluating glucose consumption, ATP synthesis, oxygen consumption rate, P/O ratio, ATP/AMP ratio, lactate release, lactate dehydrogenase activity, NAD + /NADH ratio and pyruvate dehydrogenase activity in parental and drug resistant melanoma cells. The intracellular oxidative state was analyzed in terms of reactive oxygen species production, glutathione levels and NADPH/NADP + ratio. In addition, a principal component analysis was conducted in order to identify the variables responsible for the acquisition of targeted therapy resistance., Results: Collectively, our results demonstrate, for the first time in patient-derived melanoma cells, that the rewiring of oxidative phosphorylation and the maintenance of pyruvate dehydrogenase activity and of high glutathione levels contribute to trigger the onset of PLX4032 resistance., Conclusion: Therefore, it is possible to hypothesize that inhibitors of glutathione biosynthesis and/or pyruvate dehydrogenase activity could be used in combination with PLX4032 to overcome drug resistance of BRAF-mutated melanoma patients. However, the identification of new adjuvant targets related to drug-induced metabolic reprogramming could be crucial to counteract the failure of targeted therapy in metastatic melanoma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Garbarino, Valenti, Monteleone, Pietra, Mingari, Benzi, Bruzzone, Ravera, Leardi, Farinini, Vernazza, Grottoli, Marengo and Domenicotti.)

Intraocular pressure (IOP) is considered an important modifiable risk factor for glaucoma, which is known as the second leading cause of blindness worldwide. However, lowering the IOP is not always sufficient to preserve vision due to other non-IOP-dependent mechanisms being involved. To improve outcomes, adjunctive therapies with IOP-independent targets are required. To date, no studies have shown the effect of citicoline on the trabecular meshwork (TM), even though it is known to possess neuroprotective/enhancement properties and multifactorial mechanisms of action. Given that reactive oxygen species seem to be involved in glaucomatous cascade, in this present study, an advanced millifluidic in vitro model was used to evaluate if citicoline could exert a valid TM protection against oxidative stress. To this end, the cellular behavior, in terms of viability, apoptosis, mitochondrial state, senescence and pro-inflammatory cytokines, on 3D human TM cells, treated either with H 2 O 2 alone or cotreated with citicoline, was analyzed. Our preliminary in vitro results suggest a counteracting effect of citicoline eye drops against oxidative stress on TM cells, though further studies are necessary to explore citicoline's potential as a TM-target therapy.

Alveolar macrophages are the first line of defence against detrimental inhaled stimuli. To date, no comparative data have been obtained on the inflammatory response induced by different carcinogenic mineral fibres in the three main macrophage phenotypes: M0 (non-activated), M1 (pro-inflammatory) and M2 (alternatively activated). To gain new insights into the different toxicity mechanisms of carcinogenic mineral fibres, the acute effects of fibrous erionite, crocidolite and chrysotile in the three phenotypes obtained by THP-1 monocyte differentiation were investigated. The three mineral fibres apparently act by different toxicity mechanisms. Crocidolite seems to exert its toxic effects mostly as a result of its biodurability, ROS and cytokine production and DNA damage. Chrysotile, due to its low biodurability, displays toxic effects related to the release of toxic metals and the production of ROS and cytokines. Other mechanisms are involved in explaining the toxicity of biodurable fibrous erionite, which induces lower ROS and toxic metal release but exhibits a cation-exchange capacity able to alter the intracellular homeostasis of important cations. Concerning the differences among the three macrophage phenotypes, similar behaviour in the production of pro-inflammatory mediators was observed. The M2 phenotype, although known as a cell type recruited to mitigate the inflammatory state, in the case of asbestos fibres and erionite, serves to support the process by supplying pro-inflammatory mediators.

Primary Open-Angle Glaucoma (POAG) is a neurodegenerative disease, and its clinical outcomes lead to visual field constriction and blindness. POAG's etiology is very complex and its pathogenesis is mainly explained through both mechanical and vascular theories. The trabecular meshwork (TM), the most sensitive tissue of the eye anterior segment to oxidative stress (OS), is the main tissue involved in early-stage POAG, characterized by an increase in pressure. Preclinical assessments of neuroprotective drugs on animal models have not always shown correspondence with human clinical studies. In addition, intra-ocular pressure management after a glaucoma diagnosis does not always prevent blindness. Recently, we have been developing an innovative in vitro 3Dadvanced human trabecular cell model on a millifluidicplatform as a tool to improve glaucoma studies. Herein, we analyze the effects of prolonged increased pressure alone and, in association with OS, on such in vitro platform. Moreover, we verify whethersuch damaged TM triggers apoptosis on neuron-like cells. The preliminary results show that TM cells are less sensitive to pressure elevation than OS, and OS-damaging effects were worsened by the pressure increase. The stressed TM releases harmful signals, which increase apoptosis stimuli on neuron-like cells, suggesting its pivotal role in the glaucoma cascade.

Retinal ganglion cells (RGCs) are a population of neurons of the central nervous system (CNS) extending with their soma to the inner retina and with their axons to the optic nerve. Glaucoma represents a group of neurodegenerative diseases where the slow progressive death of RGCs results in a permanent loss of vision. To date, although Intra Ocular Pressure (IOP) is considered the main therapeutic target, the precise mechanisms by which RGCs die in glaucoma have not yet been clarified. In fact, Primary Open Angle Glaucoma (POAG), which is the most common glaucoma form, also occurs without elevated IOP. This present review provides a summary of some pathological conditions, i.e., axonal transport blockade, glutamate excitotoxicity and changes in pro-inflammatory cytokines along the RGC projection, all involved in the glaucoma cascade. Moreover, neuro-protective therapeutic approaches, which aim to improve RGC degeneration, have also been taken into consideration.

Cisplatin is a first-choice chemotherapeutic agent used to treat solid tumors even though the onset of multi-drug resistance and the time-dose side-effects impair its mono-therapeutic application. Therefore, new drug-delivery approaches, based on nanomedicine strategies, are needed to enhance its therapeutic potential in favor of a dose-reduction of cisplatin. Polyunsaturated fatty acids and their metabolism-derived intermediates, as well as lipid peroxidation end-products, are used as adjuvants to improve the effectiveness of chemotherapy. Lipid hydroperoxides, derived from the oxidation of edible oils, can contribute to cell death, generating breakdown products (e.g., reactive aldehydes). In this regard, the aim of this present study was to evaluate an invitro combinatory strategy between a lecithin-based nanoemulsion system of K600, a patented mixture of peroxidated oil and peroxidated cholesterol, and cisplatin on DLD1 human adenocarcinoma cells. Our findings showed that nanoemulsions, acting in synergy with cisplatin, improve cisplatin bioactivity, in terms of enhancing its anti-cancer activity, towards DLD1 cells. Indeed, this combination approach, whilst maintaining cisplatin at low concentrations, induces a significant reduction in DLD1 cell viability, an increase in pro-apoptotic markers, and genotoxic damage. Therefore, K600 nanoemulsions as an efficient targeted delivery system of cisplatin allow for the reduction in the chemotherapeutic agent doses.

Glaucoma is a multifactorial syndrome in which the development of pro-apoptotic signals are the causes for retinal ganglion cell (RGC) loss. Most of the research progress in the glaucoma field have been based on experimentally inducible glaucoma animal models, which provided results about RGC loss after either the crash of the optic nerve or IOP elevation. In addition, there are genetically modified mouse models (DBA/2J), which make the study of hereditary forms of glaucoma possible. However, these approaches have not been able to identify all the molecular mechanisms characterizing glaucoma, possibly due to the disadvantages and limits related to the use of animals. In fact, the results obtained with small animals (i.e., rodents), which are the most commonly used, are often not aligned with human conditions due to their low degree of similarity with the human eye anatomy. Although the results obtained from non-human primates are in line with human conditions, they are little used for the study of glaucoma and its outcomes at cellular level due to their costs and their poor ease of handling. In this regard, according to at least two of the 3Rs principles, there is a need for reliable human-based in vitro models to better clarify the mechanisms involved in disease progression, and possibly to broaden the scope of the results so far obtained with animal models. The proper selection of an in vitro model with a "closer to in vivo " microenvironment and structure, for instance, allows for the identification of the biomarkers involved in the early stages of glaucoma and contributes to the development of new therapeutic approaches. This review summarizes the most recent findings in the glaucoma field through the use of human two- and three-dimensional cultures. In particular, it focuses on the role of the scaffold and the use of bioreactors in preserving the physiological relevance of in vivo conditions of the human trabecular meshwork cells in three-dimensional cultures. Moreover, data from these studies also highlight the pivotal role of oxidative stress in promoting the production of trabecular meshwork-derived pro-apoptotic signals, which are one of the first marks of trabecular meshwork damage. The resulting loss of barrier function, increase of intraocular pressure, as well the promotion of neuroinflammation and neurodegeneration are listed as the main features of glaucoma. Therefore, a better understanding of the first molecular events, which trigger the glaucoma cascade, allows the identification of new targets for an early neuroprotective therapeutic approach., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Tirendi, Saccà, Vernazza, Traverso, Bassi and Izzotti.)

Polyphenols, with anti-oxidant properties, counteract oxidative stress effects. Increasing evidence has found oxidative stressto be the main risk factor for trabecular meshwork (TM) damage, leading to high-tension glaucoma. Topical anti-oxidants could represent a new target for glaucoma treatment. Our aim is to investigate the protective mechanisms on a human TM culture of a patented polyphenol and fatty acid (iTRAB ® )formulation in response to oxidative stress using an advanced invitromodel consisting of 3D-human TM cells, embedded in a natural hydrogel, and a milli-scaled multi-organ device model for constantdynamic conditions. The 3D-human TM cells(3D-HTMCs) were treated daily with 500 µM H 2 O 2 or 500 µM H 2 O 2 and 0.15% iTRAB ® (m/v) for 72 h, and molecular differences in the intracellular reactive oxygen species (iROS), state of the cells, activation of the apoptosis pathway and NF-kB and the expression ofinflammatory and fibrotic markers wereanalyzed at different time-points.Concomitant exposure significantly reduced iROS and restored TM viability, iTRAB ® having a significant inhibitory effect on the apoptotic pathway, activation of NF-κB, induction of pro-inflammatory (IL-1α, IL-1ß and TNFα) and pro-fibrotic (TGFβ) cytokines and the matrix metalloproteinase expressions. It is clear that this specific anti-oxidant provides a valid TM protection, suggesting iTRAB ® could be an adjuvant therapy in primary open-angle glaucoma (POAG).

Primary open-angle glaucoma (POAG) is the second leading cause of irreversible blindness worldwide. Increasing evidence suggests oxidative damage and immune response defects are key factors contributing to glaucoma onset. Indeed, both the failure of the trabecular meshwork tissue in the conventional outflow pathway and the neuroinflammation process, which drives the neurodegeneration, seem to be linked to the age-related over-production of free radicals (i.e., mitochondrial dysfunction) and to oxidative stress-linked immunostimulatory signaling. Several previous studies have described a wide range of oxidative stress-related makers which are found in glaucomatous patients, including low levels of antioxidant defences, dysfunction/activation of glial cells, the activation of the NF-κB pathway and the up-regulation of pro-inflammatory cytokines, and so on. However, the intraocular pressure is still currently the only risk factor modifiable by medication or glaucoma surgery. This present review aims to summarize the multiple cellular processes, which promote different risk factors in glaucoma including aging, oxidative stress, trabecular meshwork defects, glial activation response, neurodegenerative insults, and the altered regulation of immune response., Competing Interests: The authors declare no conflict of interest

In this manuscript, which appeared in ALTEX 37, 265-274 (doi: 10.14573/altex.1909262), the affiliation of Stefania Vernazza should read: Stefania Vernazza 5# 5 IRCCS-Fondazione Bietti, Rome, Italy and the address for correspondence should read: Stefania Vernazza, PhD, IRCCS, Fondazione Bietti via Livenza 3, 00198 Rome, Italy (stefania.vernazza@yahoo.it).

Glaucoma is the second leading cause of blindness worldwide. Currently, glaucoma treatments aim to lower intraocular pressure by decreasing aqueous humor production or increasing aqueous humor outflow through pharmacological approaches or trabeculectomy. The lack of an effective cure requires new therapeutic strategies. We compared the bio­logical responses of a three-dimensional trabecular meshwork model with or without perfusion bioreactor technology to better understand the early molecular changes induced by prolonged oxidative stress conditions induced by repeated daily peroxide exposure. We used standard 3D cultures of trabecular meshwork cells in Matrigel cultured under either static and dynamic conditions for one week. We studied changes in F-actin expression and organization in the cells, cellular metabolic activity, proinflammatory gene expression, expression of pro- and anti-apoptotic proteins, PARP-1 cleavage, and NFκB activation in the model. We demonstrate that the dynamic conditions improve the adaptive behavior of 3D trabecular meshwork cultures to chronic oxidative stress via offsetting pathway activation.

A physiologically relevant in vitro human-based model could be the 'gold standard' to clarify the pathological steps involved in glaucoma onset. In this regard, human 3D cultures may represent an excellent starting point to achieve this goal. Indeed, the 3D matrix allows to re-create the in vivo-like tissue architecture, maintaining its functionality and cellular behaviour, compared to the 2D model. Thus, we propose a comparison between the 2D and 3D in vitro models of human trabecular meshwork cells in terms of cellular responses after chronic stress exposure. Our results showed that 3D-cells are more sensitive to intracellular reactive oxidative specie production induced by hydrogen peroxide treatment, compared to 2D cultures. Additionally, in 3D cultures a more accurate regulation of the apoptosis trigger and cell adaptation mechanisms was detected than in 2D models. In line with these findings, the 3D-HTMC model shows the ability to better mimic the in vivo cell behaviour in adaptive responses to chronic oxidative stress than 2D., Competing Interests: The authors have declared that no competing interests exist.

Alterations in the control of apoptotic processes were observed in cells during space flight or under simulated microgravity, the latter obtained with the 3D-Random Positioning Machine (3D-RPM). Usually the proteins Bax and Bcl-2, act as pro- or anti-apoptotic regulators. Here we investigated the effects of simulated microgravity obtained by the 3D-RPM on cell viability, localization and expression of Bax and Bcl-2 in cultures of glial cancerous cells. We observed for the first time a transient cytoplasmic/nuclear translocation of Bax and Bcl-2 triggered by changing gravity vector. Bax translocates into the nucleus after 1 h, is present simultaneously in the cytoplasm after 6 h and comes back to the cytoplasm after 24 h. Bcl-2 translocate into the nucleus only after 6 h and comes back to the cytoplasm after 24 h. Physiological meaning, on the regulation of apoptotic event and possible applicative outcomes of such finding are discussed.