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AIM AND OBJECTIVE The objective of this study was to monitor the pH stability over a period of 6 months of two-bottle self-etching (SE) primers. MATERIALS AND METHODS Four commercially available two-bottle SE primer solutions, Adhese SE (Ivoclar Vivadent; Schaan, Liechtenstein), Clearfil SE (Kuraray; Tokyo, Japan), Contax Primer (DMG; Hamburg, Germany), and Enabond SE (Micerium; Avegno (Ge), Italy), containing water as the main solvent were selected. The pH values during a 6-month storage period at intermittent temperature were daily measured by means of a microprocessor-based pH/temperature meter and hence recorded, tabulated, and analyzed. RESULTS The mean values of overall pH measurements and standard deviation for Adhese SE, Clearfil SE, Contax Primer, and Enabond SE are, respectively, 1.70 (±0.01), 1.72 (±0.01), 1.20 (±0.01), 1.80 (±0.01). One-way ANOVA did not detect any significant change of the mean pH values of all measurements over time. CONCLUSION The conventional 6-months storage did not affect the pH values of the water-containing primers of two-bottle SE adhesives. CLINICAL SIGNIFICANCE This study provides evidence that conventional storage under adequate conditions did not affect the pH values of the water-containing primers of two-bottle SE adhesives with different monomer formulations. Clinicians may rely on the known pH value of the selected SE adhesive during shelf-life.

Tailoring the cell organelles and thus changing cell homeostatic behavior has permitted the discovery of fascinating metabolic features enabling enhanced viability, differentiation, or quenching inflammation. Recently, photobiomodulation (PBM) has been accredited as an effective cell manipulation technique with promising therapeutic potential. In this prospective, in vitro results revealed that 808-nm laser light emitted by a hand-piece with a flat-top profile at an irradiation set up of 60 J/cm2 (1 W, 1 W/cm2; 60 s, continuous wave) regulates bone marrow stromal cell (BMSC) differentiation toward osteogenesis. Considering the importance of actin cytoskeleton reorganization, which controls a range of cell metabolic activities, comprising shape change, proliferation and differentiation, the aim of the current work is to assess whether PBM therapy, using a flat-top hand-piece at higher-fluence irradiation on BMSCs, is able to switch photon signals into the stimulation of biochemical/differentiating pathways involving key activators that regulate de novo actin polymerization. Namely, for the first time, we unearthed the role of the flat-top hand-piece at higher-fluence irradiation on cytoskeletal characteristics of BMSCs. These novel findings meet the needs of novel therapeutically protocols provided by laser treatment and the manipulation of BMSCs as anti-inflammatory, osteo-inductive platforms.

Photobiomodulation (PBM) consists of a photon energy transfer to the cell, employing non-ionizing light sources belonging to the visible and infrared spectrum. PBM acts on some intrinsic properties of molecules, energizing them through specific light wavelengths. During the evolution of life, semiconducting minerals were energized by sun radiation. The molecules that followed became photoacceptors and were expressed into the first proto-cells and prokaryote membranes. Afterward, the components of the mitochondria electron transport chain influenced the eukaryotic cell physiology. Therefore, although many organisms have not utilized light as an energy source, many of the molecules involved in their physiology have retained their primordial photoacceptive properties. Thus, in this review, we discuss how PBM can affect the oral microbiota through photo-energization and the non-thermal effect of light on photoacceptors (i.e., cytochromes, flavins, and iron-proteins). Sometimes, the interaction of photons with pigments of an endogenous nature is followed by thermal or photodynamic-like effects. However, the preliminary data do not allow determining reliable therapies but stress the need for further knowledge on light-bacteria interactions and microbiota management in the health and illness of patients through PBM.

The tremendous therapeutic potential of photobiomodulation therapy in different branches of medicine has been described in the literature. One of the molecular mechanisms for this treatment implicates the mitochondrial enzyme, cytochrome C oxidase. However, the efficacy and consistency of clinical outcomes with photobiomodulation treatments has been fiercely debated. This work was motivated by this need to improve photobiomodulation devices and delivery approaches. We designed a novel hand-piece with a flat-top beam profile of irradiation. We compared the beam profile versus a standard hand-piece and a fibre probe. We utilized isolated mitochondria and performed treatments at various spots within the beam, namely, the centre, left and right edge. We examined mitochondrial activity by assessing ATP synthesis with the luciferin/luciferase chemiluminescent method as a primary endpoint, while mitochondrial damage was assessed as the secondary endpoint. We observed a uniform distribution of the power density with the flat-top prototype compared to a wide Gaussian beam profile with the standard fibre and standard hand-piece. We noted increased production of ATP in the centre of all three beams with respect to the non-treated controls (p &lt, 0.05). Both the fibre and standard hand-piece demonstrated less increase in ATP synthesis at the edges than the centre (p &lt, 0.05). In contrast, ATP synthesis was increased homogenously in the flat-top handpiece, both in the centre and the edges of the beam. Fibre, standard hand-piece and the flat-top hand-piece prototype have discrete beam distribution characteristics. This significantly affected the mitochondrial activity with respect to their position within the treated areas. Flat-top hand-piece enhances the uniformity of photobiomodulation treatments and can improve the rigour and reproducibility of PBM clinical outcomes.

Background: bone tissue regeneration remains a current challenge. A growing body of evidence shows that mitochondrial dysfunction impairs osteogenesis and that this organelle may be the target for new therapeutic options. Current literature illustrates that red and near-infrared light can affect the key cellular pathways of all life forms through interactions with photoacceptors within the cells’ mitochondria. The current study aims to provide an understanding of the mechanisms by which photobiomodulation (PBM) by 900-nm wavelengths can induce in vitro molecular changes in pre-osteoblasts. Methods: The PubMed, Scopus, Cochrane, and Scholar databases were used. The manuscripts included in the narrative review were selected according to inclusion and exclusion criteria. The new experimental set-up was based on irradiation with a 980-nm laser and a hand-piece with a standard Gaussian and flat-top beam profile. MC3T3-E1 pre-osteoblasts were irradiated at 0.75, 0.45, and 0.20 W in continuous-wave emission mode for 60 seconds (spot-size 1 cm2) and allowed to generate a power density of 0.75, 0.45, and 0.20 W/cm2 and a fluence of 45, 27, and 12 J/cm2, respectively. The frequency of irradiation was once, three times (alternate days), or five times (every day) per week for two consecutive weeks. Differentiation, proliferation, and cell viability and their markers were investigated by immunoblotting, immunolabelling, fluorescein-FragELTM-DNA, Hoechst staining, and metabolic activity assays. Results and conclusions: The 980-nm wavelength can photobiomodulate the pre-osteoblasts, regulating their metabolic schedule. The cellular signal activated by 45 J/cm2, 0.75 W and 0.75 W/cm2 consist of the PI3K/Akt/Bcl-2 pathway, differentiation markers were not affected, nor do other parameters seem to stimulate the cells. Our previous and present data consistently support the window effect of 980 nm, which has also been described in extracted mitochondria, through activation of signalling PI3K/Akt/Bcl-2 and cyclin family, while the Wnt and Smads 2/3-β-catenin pathway was induced by 55 J/cm2, 0.9 W and 0.9 W/cm2.

Background: Due to the lack of data on bone-to-graft contact (BGC) over time in the various regions within the subantral space of the augmented sinus floor, the present study aimed to evaluate the osteoconductivity of deproteinized bovine bone mineral (DBBM) with granules of different sizes applied in maxillary sinus floor elevation. Methods: A maxillary sinus augmentation was performed bilaterally in 18 rabbits using DBBM with particle dimensions of either 0.125–1.0 mm or 1–2 mm. The antrostomy was covered using a collagen barrier. The animals were euthanized in groups of six after 2, 4, and 8 weeks of healing. MicroCT and histological analyses were performed. Results: After 2 weeks of healing, BGC was 10.9% and 11.9% for the small and large granule sites, respectively. After 8 weeks of healing, the BGC increased to 65% and 62% at the small and large granule sites, respectively. The highest values were located close to the bony walls and the bony window. New bone content developed between 2 and 8 weeks from 7.0% to 27.6% and from 6.1% to 27.6% at the small and large granule sites, respectively. Conclusions: Similar outcomes in osteoconductivity and bone formation were found at both small and large DBBM granule sites.

Background: Injury of the trigeminal nerve in oral and maxillofacial surgery can occur. Schwann cell mitochondria are regulators in the development, maintenance and regeneration of peripheral nerve axons. Evidence shows that after the nerve injury, mitochondrial bioenergetic dysfunction occurs and is associated with pain, neuropathy and nerve regeneration deficit. A challenge for research is to individuate new therapies able to normalise mitochondrial and energetic metabolism to aid nerve recovery after damage. Photobiomodulation therapy can be an interesting candidate, because it is a technique involving cell manipulation through the photonic energy of a non-ionising light source (visible and NIR light), which produces a nonthermal therapeutic effect on the stressed tissue. Methods: The review was based on the following questions: (1) Can photo-biomodulation by red and NIR light affect mitochondrial bioenergetics? (2) Can photobiomodulation support damage to the trigeminal nerve branches? (preclinical and clinical studies), and, if yes, (3) What is the best photobiomodulatory therapy for the recovery of the trigeminal nerve branches? The papers were searched using the PubMed, Scopus and Cochrane databases. This review followed the ARRIVE-2.0, PRISMA and Cochrane RoB-2 guidelines. Results and conclusions: The reliability of photobiomodulatory event strongly bases on biological and physical-chemical evidence. Its principal player is the mitochondrion, whether its cytochromes are directly involved as a photoacceptor or indirectly through a vibrational and energetic variation of bound water: water as the photoacceptor. The 808-nm and 100 J/cm2 (0.07 W; 2.5 W/cm2; pulsed 50 Hz; 27 J per point; 80 s) on rats and 800-nm and 0.2 W/cm2 (0.2 W; 12 J/cm2; 12 J per point; 60 s, CW) on humans resulted as trustworthy therapies, which could be supported by extensive studies.

Background: the aphtha is one of the most common oral mucosal ulcerations and presents as a painful punched-out sore. Systemic and topical medications are used to reduce inflammation and pain and to support the natural period of remission. Alternative treatment modalities have been requested to relieve pain and improve its healing. In this regard, photobiomodulation, which is a manipulation of cells’ metabolism through an energy transfer by light sources of non-ablative or thermal intensity, could support aphtha management. The predictor variable of our research was the photobiomodulation through higher energy and power irradiated through a handpiece with a flat-top beam profile. The primary end point was the complete healing of the aphtha, defined as the time from the irradiation to the complete recovery. The secondary end point was pain relief, evaluated daily through the visual analogue scale (VAS), from the irradiation to 24 and 48 h after. Methods: a randomized, double-blind, controlled trial was conducted according to the CONSORT guideline. Irradiation was performed through an 808-nm diode laser with flat-top handpiece, and 1 W, 1 W/cm2, 60 J, 60 J/cm2 for 60 s on a spot-size area of 1 cm2. Time of complete healing and pain evaluation by VAS scale were evaluated. Results: between 1 January, 2020 and 1 March, 2021, 126 patients were screened for the study at the Department of Surgical and Diagnostic Sciences, University of Genoa, Italy. Sixty patients were randomly assigned (30 in the photobiomodulation group and 30 in the placebo group). Patients of the photobiomodulation group experienced complete healing in an average time of 8.13 days ± 1.69 (min 5–max 10 days), while for the placebo group the average time extended to 30.76 ± 4.63 days (min 25–max 42 days). Patients of the photobiomodulation therapy group experienced a statistically significant reduction in pain and discomfort 24 and 48 h after treatment (p &lt, 0.05), the reduction was statistically higher (p &lt, 0.05) 48 h after treatment compared to 24 h after. Conclusions: photobiomodulation at the parameters and modality of irradiation proposed accelerates the healing recovery and reduces pain compared to the patients treated with the placebo.

Photobiomodulation with 808 nm laser light electively stimulates Complexes III and IV of the mitochondrial respiratory chain, while Complexes I and II are not affected. At the wavelength of 1064 nm, Complexes I, III, and IV are excited, while Complex II and some mitochondrial matrix enzymes seem to be not receptive to photons at that wavelength. Complex IV was also activated by 633 nm. The mechanism of action of wavelengths in the range 900–1000 nm on mitochondria is less understood or not described. Oxidative stress from reactive oxygen species (ROS) generated by mitochondrial activity is an inescapable consequence of aerobic metabolism. The antioxidant enzyme system for ROS scavenging can keep them under control. However, alterations in mitochondrial activity can cause an increment of ROS production. ROS and ATP can play a role in cell death, cell proliferation, and cell cycle arrest. In our work, bovine liver isolated mitochondria were irradiated for 60 sec, in continuous wave mode with 980 nm and powers from 0.1 to 1.4 W (0.1 W increment at every step) to generate energies from 6 to 84 J, fluences from 7.7 to 107.7 J/cm2, power densities from 0.13 to 1.79 W/cm2, and spot size 0.78 cm2. The control was equal to 0 W. The activity of the mitochondria’s complexes, Krebs cycle enzymes, ATP production, oxygen consumption, generation of ROS, and oxidative stress were detected. Lower powers (0.1–0.2 W) showed an inhibitory effect; those that were intermediate (0.3–0.7 W) did not display an effect, and the higher powers (0.8–1.1 W) induced an increment of ATP synthesis. Increasing the power (1.2–1.4 W) recovered the ATP production to the control level. The interaction occurred on Complexes III and IV, as well as ATP production and oxygen consumption. Results showed that 0.1 W uncoupled the respiratory chain and induced higher oxidative stress and drastic inhibition of ATP production. Conversely, 0.8 W kept mitochondria coupled and induced an increase of ATP production by increments of Complex III and IV activities. An augmentation of oxidative stress was also observed, probably as a consequence of the increased oxygen consumption and mitochondrial isolation experimental conditions. No effect was observed using 0.5 W, and no effect was observed on the enzymes of the Krebs cycle.

After 50 years of studies on photobiomodulation (PBM), there is still so much to investigate to understand the laser light-nonplant cells interactions. The current scientific knowledge allows to say that the phenomena induced by PBM are based on cellular pathways that are the key points of cell life. The mitochondria chromophores, also present on the bacterial membrane, the calcium channels, ion that regulates the life-and-death cellular processes, as well as the TRP family, whose genes have been found in protozoa and suggest that its basic mechanism evolved long before the appearance of animals, seem to be elective targets in photobiomodulatory events by wavelengths from 600 up to 980 nm. The ambiguous resulting cellular communication way, mediated by ATP, ROS and/or calcium, leads to cell manipulation, which modifies its metabolism and whose response connects all life-forms from bacteria to vertebrates. Because of the Giano-Bifronte features of ROS and calcium, as well as the fine balance of energetic mitochondrial processes, whose alteration is responsible for several diseases, the PBM can show unpredictable results and it requires scrupulous approach to avoid cellular damages. However, when carefully applied, PBM is able to improve nonhealthy cell's responses and represents a reliable support in human and veterinary medicine.

The p62 (also named sequestosome1/SQSTM1) is multidomain and multifunctional protein associated with several physiological and pathological conditions. A number of studies evidenced an involvement of p62 on the disruptive bone scenarios due to its participation in the inflammatory/osteoclastogenic pathways. However, so far, information regarding the function of p62 in the fine-tuned processes underpinning the bone physiology are not well-defined and are sometime discordant. We, previously, demonstrated that the intramuscular administration of a plasmid coding for p62 was able to contrast bone loss in a mouse model of osteopenia. Here, in vitro findings showed that the p62 overexpression in murine osteoblasts precursors enhanced their maturation while the p62 depletion by a specific siRNA, decreased osteoblasts differentiation. Consistently, the activity of osteoblasts from p62-/- mice was reduced compared with wild-type. Also, morphometric analyses of bone from p62 knockout mice revealed a pathological phenotype characterized by a lower turnover that could be explained by the poor Runx2 protein synthesis in absence of p62. Furthermore, we demonstrated that the parathyroid hormone (PTH) regulates p62 expression and that the osteogenic effects of this hormone were totally abrogated in osteoblasts from p62-deficient mice. Therefore, these findings, for the first time, highlight the important role of p62 both for the basal and for PTH-stimulated bone remodeling.

Background A large number of systemic diseases can be linked to oral candida pathogenicity. The global trend of invasive candidiasis has increased progressively and is often accentuated by increasing Candida albicans resistance to the most common antifungal medications. Photodynamic therapy (PDT) is a promising therapeutic approach for oral microbial infections. A new formulation of 5-aminolevulinic acid (5%ALA) in a thermosetting gel (t) (5%ALA-PTt) was patented and recently has become available on the market. However, its antimicrobial properties, whether mediated or not by PDT, are not yet known. In this work we characterised them. Methods We isolated a strain of C. albicans from plaques on the oral mucus membrane of an infected patient. Colonies of this strain were exposed for 1 24 h, to 5%ALA-PTt, 5%ALA-PTt buffered to pH 6.5 (the pH of the oral mucosa) (5%ALA-PTtb) or not exposed (control). The 1 h-exposed samples were also irradiated at a wavelength of 630 nm with 0.14 watts (W) and 0.37 W/cm2 for 7 min at a distance of Results and conclusion The 5% ALA-PTt preparation was shown to be effective in reducing the growth of biofilm and inoculum of C. albicans. This effect seems to be linked to the intrinsic characteristics of 5%ALA-TPt, such acidic pH and the induction of free radical production. This outcome was significantly enhanced by the effect of PDT at relatively short incubation and irradiation times, which resulted in growth inhibition of both treated biofilm and inoculum by ∼80% and ∼95%, respectively.

Photobiomodulation relies on the transfer of energy from incident photons to a cell photoacceptor. For many years the concept of photobiomodulation and its outcome has been based upon a belief that the sole receptor within the cell was the mitochondrion. Recently, it has become apparent that there are other photoacceptors operating in different regions of the electromagnetic spectrum. Alternative photoacceptors would appear to be water and mechanisms regulating calcium homeostasis, despite a direct effect of laser photonic energy on intracellular calcium concentration outwith mitochondrial activity or influence, have not been clearly demonstrated. Therefore, to increase the knowledge of intracellular‑calcium and laser photon interaction, as well as to demonstrate differences in irradiation profiles with modern hand-pieces, we tested and compared the photobiomodulatory effect of 808 nm and 980 nm diode laser light by low- and higher-energy (60s, 100 mW/cm2, 100 mW/cm2, 500 mW/cm2, 1000 mW/cm2, 1500 mW/cm2, 2000 mW/cm2) irradiated with a “standard” (Gaussian fluence distribution) hand-piece or with a “flat-top” (uniform fluence) hand-piece. For this purpose, we used the eukaryote unicellular-model Dictyostelium discoideum. The 808 nm and 980 nm infrared laser light, at the energy tested directly affect the stored Ca2+ homeostasis, independent of the mitochondrial respiratory chain activities. From an organism perspective, the effect on Ca2+-dependent signal transduction as the regulator of spore germination in Dictyostelium, demonstrates how a cell can respond quickly to the correct laser photonic stimulus through a different cellular pathway than the known light-chromophore(mitochondria) interaction. Additionally, both hand-piece designs tested were able to photobiomodulate the D. discoideum cell; however, the hand-piece with a flat-top profile, through uniform fluence levels allows more effective and reproducible effects.

Photobiomodulation (PBM) is a non-plant-cell manipulation through a transfer of energy by means of light sources at the non-ablative or thermal intensity. Authors showed that cytochrome-c-oxidase (complex IV) is the specific chromophore's target of PBM at the red (600-700 nm) and NIR (760-900 nm) wavelength regions. Recently, it was suggested that the infrared region of the spectrum could influence other chromospheres, despite the interaction by wavelengths higher than 900 nm with mitochondrial chromophores was not clearly demonstrated. We characterized the interaction between mitochondria respiratory chain, malate dehydrogenase, a key enzyme of Krebs cycle, and 3-hydroxyacyl-CoA dehydrogenase, an enzyme involved in the β-oxidation (two mitochondrial matrix enzymes) with the 1064 nm Nd:YAG (100mps and 10 Hz frequency mode) irradiated at the average power density of 0.50, 0.75, 1.00, 1.25 and 1.50 W/cm2 to generate the respective fluences of 30, 45, 60, 75 and 90 J/cm2 . Our results show the effect of laser light on the transmembrane mitochondrial complexes I, III, IV and V (adenosine triphosphate synthase) (window effects), but not on the extrinsic mitochondrial membrane complex II and mitochondria matrix enzymes. The effect is not due to macroscopical thermal change. An interaction of this wavelength with the Fe-S proteins and Cu-centers of respiratory complexes and with the water molecules could be supposed.

Background Any successful endodontic therapy requires elimination of the endodontic biofilms through meticulous root canal disinfection methods. Sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid (EDTA) are the most common effective irrigants, in removing smear-layer from the coronal and middle thirds of the dental canals but reduced considerably towards the apical one third. In recent years, newly alternative treatment modalities have been proposed, including high-power lasers and antimicrobial photodynamic therapy (aPDT). Our work was conducted to evalaute the outcome of root canal disinfection in relation to the efficacy of various treatment modalities. Furthermore, every effort was made to present an overview of the aPDT outcomes, as a model for this application, and to propose laser parameters protocol with positive results. Methods The electronic databases PubMed was searched from January 2013- January 2019. Our inclusive criteria based on laser therapy applications, as a model for root canal disinfection. The search terms utilised various combinations as follows: photodynamic therapy or antimicrobial photodynamic therapy or photoactivated disinfection or light activated disinfection or laser activated disinfection or laser therapy, and endodontic. Results The results of this systematic review concluded that the effectiveness of aPDT and various laser wavelengths protocols, in removing endodontic biofilms from infected root canals, remains unattainable. Conclusions Study concluded that the combination of aPDT with antimicrobial irrigants could provide a synergetic effect. However, due to the heterogeneity of the selected studies and their limitations, in terms of lack of standardised protocol or discrepancy in the methodology, authors suggest further validated approaches to achieve optimal outcomes.

Compounds known as chemical mediators, including acetylcholine, have been found not only in humans and animals, but also in living organisms, like protozoa, which lack nervous system. In Paramecium primaurelia has been described a cholinergic system, which is proven to play an important role in cell-cell interactions during its developmental cycle. In our work we investigated the effects of exposure to simulated microgravity (3D Random Positioning Machine, 56 rpm, 10-6 g) on the cholinesterase activity of the eukaryote unicellular-organism alternative-model P. primaurelia. Our results show that the exposure of P. primaurelia to microgravity for 6 h, 24 h, 48 h affects the localization and the amount of cholinesterase activity compared to cells grown under Earth gravity conditions (1 g). However, these effects are transient since P. primaurelia restores its normal cholinesterase activity after 72 h under microgravity conditions, as well as cells exposed up to 72 h to microgravity and then placed under terrestrial gravity for 48 h.

Photobiomodulation of cells using near-infrared (NIR) monochromatic light can affect cell functions such as proliferation, viability, and metabolism in a range of cell types. Evidence for the effects of near-infrared light on endothelial cells has been reported, but the studies were mainly performed using VIS light emitted by low-energy lasers, because NIR wavelengths seemed negatively stimulate these cells. Cell viability, free radical-induced oxidative stress, NF-κB activation, nitric oxide release, mitochondrial respiration, and wound healing repair were assessed in human endothelial cells (HECV) irradiated with 808-nm diode laser light (laser setup = 1 W/cm2, 60 s, 60 J/cm2, CW vs measured energy parameter = 0.95 W/cm2, 60 s, 57 J/cm2, mode CW) emitted by an handpiece with flat-top profile. No difference in viability was detected between controls and HECV cells irradiated with 808-nm diode laser light for 60 s. Irradiated cells demonstrated higher proliferation rate and increased migration ability associated to moderate increase in ROS production without a significant increase in oxidative stress and oxidative stress-activated processes. Near-infrared light stimulated mitochondrial oxygen consumption and ATP synthesis in HECV cells. Short near-infrared irradiation did not affect viability of HECV cells, rather led to a stimulation of wound healing rate, likely sustained by ROS-mediated stimulation of mitochondrial activity. Our results demonstrating that near-infrared led to a shift from anaerobic to aerobic metabolism provide new insight into the possible molecular mechanisms by which photobiomodulation with 808-nm diode laser light protects against inflammation-induced endothelial dysfunction, seemingly promising to enhance their therapeutic properties.

The literature has supported the concept of mesenchymal stromal cells (MSCs) in bone regeneration as one of the most important applications in oro-maxillofacial reconstructions. However, the fate of the transplanted cells and their effects on the clinical outcome is still uncertain. Photobiomodulation (PBM) plays an important role in the acceleration of tissue regeneration and potential repair. The aim of this in vitro study is to evaluate the effectiveness of PBM with 808 nm diode laser therapy, using a flat-top hand-piece delivery system at a higher-fluence (64 J/cm2) irradiation (1 W, continuous-wave) on bone marrow stromal cells (BMSCs). The BMSCs of 3 old female Balb-c mice were analyzed. The cells were divided into two groups: irradiated group and control group. In the former the cells were irradiated every 24 h during 0 day (T0), 5 (T1), 10 (T2), and 15 (T3) days, whereas the control group was non-irradiated. The results have shown that the 64 J/cm2 laser irradiation has increased the Runt-related transcription factor 2 (Runx2). Runx2 is the most important early marker of osteoblast differentiation. The higher-fluence suppressed the synthesis of adipogenic transcription factor (PPARγ), the pivotal transcription factor in adipogenic differentiation. Also, the osteogenic markers such as Osterix (Osx) and alkaline phosphatase (ALP) were upregulated with an increase in the matrix mineralization. Furthermore, western blotting data demonstrated that the laser therapy has induced a statistically valid increase in the synthesis of transforming growth factor β1 (TGF-β1) but had no effects on the tumor necrosis factor α (TNFα) production. The data has statistically validated the down-regulation of the important pro-inflammatory cytokines such as interleukin IL-6, and IL-17 after 808 nm PBM exposition. An increase in anti-inflammatory cytokines such as IL-1rα and IL-10 was observed. These in vitro studies provide for first time the initial proof that the PBM of the 808 nm diode laser therapy with flat-top hand-piece delivery system at a higher-fluence irradiation of 64 J/cm2 (1 W/cm2) can modulate BMSCs differentiation in enhancing osteogenesis.

The use of pyrethroids has increased throughout the world over the past few decades, as organophosphate, carbamate and organochlorine insecticides are being phased out. Permethrin is widely used in the USA for crops treatment, at concentrations around 750 × 103 μg/L. In our study 3.6 μg/L permethrin decreases the fission-rate and the fruiting bodies formation of slime mould Dictyostelium discoideum. Whereas 3.6 × 104 μg/L kills the 100% of amoebae, showing a 24 h-LC50 = 96.6 μg/L. This concentration induces an increase in the pseudocholinesterase activity as well as in both butyrylcholinesterase and heat-shock-protein 70 presence. Our results highlight the high sensitivity of Dictyostelium to permethrin, at concentration of about 105 lesser than what used for agricultural pest control. If we match our results on 6 days of exposure, with the permethrin relatively slow permanence (30 days) in the aerobic soil, as well as the higher effect of permethrin than organophosphate, carbamate and organochlorine pesticides on D. discoideum, the damage on the dictyostelids community, by use of permethrin, is clear. Our data suggest that, if the sustainable agriculture implementation is a topic of the modern "industrial" farming, the permethrin cannot represent a reliable alternative to organochlorine, organophosphate or carbamate pesticides, in implementing Integrated Pest Management programmes.

Slime moulds live in agricultural ecosystems, where they play an important role in the soil fertilization and in the battle against crop pathogens. In an agricultural soil, the amoebae are exposed to different stress factors such as pesticides and weather conditions. The use of pesticides increased up from 0.49 kg per hectare in 1961 to 2 kg in 2004, and the global greenhouse gas emission has grown 70 % between 1970 and 2004 leading to a global fluctuation of average surface temperature. Therefore, the European Directive 2009/128/EC has led to a new approach to agriculture, with the transition from an old concept based on high use of pesticides and fossil fuels to an agriculture aware of biodiversity and health issues. We studied the effects of temperature variations and pesticides on Dictyostelium discoideum. We measured the fission rate, the ability to differentiate and the markers of stress such as the activity and presence of pseudocholinesterase and the presence of heat shock protein 70. Our results highlight how the sensitivity to zinc, aluminium, silver, copper, cadmium, mercury, diazinon and dicofol changes for a 2 °C variation from nothing/low to critical. Our work suggests considering, in future regulations, about the use of pesticides as their toxic effect on non-target organisms is strongly influenced by climate temperatures. In addition, there is a need for a new consideration of the protozoa, which takes into account recent researches about the presence in this microorganism of classical neurotransmitters that, similar to those in animals, make protozoa an innocent target of neurotoxic pesticides in the battle against the pest crops.

The development of technologies that generate environmental electromagnetic fields (EMFs) has led public opinion and the scientific community to debate upon the existence of possible effects caused by man-made EMFs on the human population and, more generally, on terrestrial ecosystems. Protozoa are known to be excellent bioassay systems in bioelectromagnetic studies because of their features that combine the reliability of in vivo results with the practicality of in vitro ones. For this reason, we examined the possible stressful effects of a 50-Hz, 300-μT extremely low-frequency electromagnetic field (ELF-EMF) on the protozoan Dictyostelium discoideum, which was used as it is included in the eight bioassay alternatives to vertebrate models for the study of human disease by the U.S. National Institutes of Health. Our results show how a 24-h exposure of D. discoideum cells to ELF-EMF can affect the net fission rate, the activity and presence of the pseudocholinesterase as well as the presence of the heat shock protein-70, while no change in the catalase and glutathione peroxidase activities was observed. However, this effect seems to be transient and all the altered parameters returned to their respective control value after a 24-h stay under dummy exposure conditions.

Studies by researchers worldwide have revealed that, even in industrialised nations, people, infants and the aged in particular, are even more exposed to neurotoxic drugs as a consequence of the increased quantity of pesticide residues in food. This phenomenon, as underlined by The Worldwatch Institute (2006) , is linked to the exponential increase in the use of these toxic compounds over the last 40 years, up from 0.49 kg per hectare in 1961 to 2 kg in 2004, with the result that these substances are found in the daily diet. Many studies have demonstrated how the assumption of pesticides in the neonatal period and early infancy can alter the development and function of the nervous, immune, endocrine and reproductive apparatuses. Moreover, the unequivocal relationship between brain tumours, infant leukemia and pesticides are well recognised. On the basis of the above information, the effects of the neurotoxic thionophosphate pesticide chlorpyrifos (CPF) have been tested, considering biomarkers of toxicity and toxicity endpoint, on the biological models Dictyostelium discoideum , Paracentrotus lividus , and NTera2 Cells, as they are compatible with the 3Rs strategy (Reduction, Replacement, and Refinement in animal experiments). Our results have revealed that developing organisms are particularly sensitive to the toxic effects of CPF.

Few studies exist to explore the potential photobiomodulation (PBM) effect of neodymium:yttrium-aluminium garnet (Nd:YAG) laser irradiation using a flat-top handpiece delivery system. In this study, we explored the photobiomodulation effect of that laser, on Paramecium primaurelia. The parameters for the different study groups were: 0.50W, 10Hz, 100msp, 30J/cm2; 0.75W, 10Hz, 100msp, 45J/cm2; 1.00W, 10Hz, 100msp, 60J/cm2; 1.25W, 10Hz, 100msp, 75J/cm2 and 1.50W, 10Hz, 100msp, 90J/cm2. Our results suggest that only the parameter 0.5W, 10Hz, 100msp, 30J/cm2 positively photobiomodulates the Paramecium cells inducing an increment in oxygen consumption, endogenous ATP synthesis and fission rate rhythm. Applying the laser energy with parameters of 1.25W, 10Hz, 100msp, 75J/cm2 and 1.50W, 10Hz, 100msp, 90J/cm2, induce adverse effect on the Paramecium cells, which protect themselves through the increase in Heat Shock Protein-70 (HSP70). The data presented in our work support our assumption that, when using appropriate parameters of irradiation, the 1064nm Nd:YAG laser with flat-top handpiece could be a valuable aid for effective clinical application of PBM.

In the originally published article, the name of the first author was incorrectly labeled. Given name is Isabelle and family name is Fini Storchi.

Although photons have been repeatedly shown to affect the functioning of the nervous system, their effects on neurotransmitter release have never been investigated. We exploited in vitro models that allow effects involving neuron-astrocyte network functioning to be detected (mouse cerebrocortical slices) and dissected these effects at cerebrocortical nerve endings and astrocyte processes. Infrared light proved able to induce glutamate release by stimulating glutamatergic nerve endings.

The notable increase in agricultural and industrial activities over the last decades has caused a considerable increase in anthropogenic waste and, consequently, the presence of pollutants in both water and sediments. For this reason, there is great interest in identifying alternative models and bioassays complying with the 3Rs strategy (aimed at Reducing, Refining and Replacing tests on vertebrate organisms in toxicological studies). Protozoa seem to be well suited to this strategy and it is widely accepted that assays with protozoa are relevant to the study of environmental modifications due to the presence of xenobiotic compounds. Recently, we detected the presence of nitric oxide synthase (NOS)-related NADPH-diaphorase activity and neuronal NOS-related molecules, immunologically recognized by the anti-rat brain NOS antibody, in a single-cell freshwater eukaryote, Paramecium primaurelia . In this work we have looked for the basal NO production in living cells of P. primaurelia using the specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA) and measuring the intracellular NO levels with image analysis. The NO production was sensitive to compounds modulating NOS activity such as: S-methyl-tiocitrulline, an NOS activity inhibitor, l -NAME, an analogue of arginine that inhibits NO production, arginine, an NOS substrate, or sodium nitroprusside, an NO donor. The NO production in P. primaurelia was also shown to be sensitive to μM concentrations of heavy metals (HgCl 2 and CdCl 2 ), or μM concentrations of pesticides (diazinon and AFD 25), thus representing a potential biomarker for environmental biomonitoring. The possible involvement of cellular Ca 2+ concentration, assayed by the fluorescent probe chlortetracycline hydrochloride, in NO production was examined after xenobiotic exposure.

Objective: Photobiomodulation is proposed as a non-linear process. Only the action of light at a low intensity and fluence is assumed to have stimulation on cells; whereas a higher light intensity and fluence generates negative effects, exhausting the cell's energy reserve as a consequence of a too strong stimulation. In our work, we detected the photobiomodulatory effect of an 808-nm higher-fluence diode laser [64 J/cm2–1 W, continuous wave (CW)] irradiated by a flat-top handpiece on mitochondria activities, such as oxygen consumption, activity of mitochondria complexes I, II, III, and IV, and cytochrome c as well as ATP synthesis. Materials and methods: The experiments are performed by standard procedure on mitochondria purified from bovine liver. Results: Our higher-fluence diode laser positively photobiomodulates the mitochondria oxygen consumption, the activity of the complexes III and IV, and the ATP production, with a P/O = 2.6. The other activities are not influenced. Conclusion: Our data...

Photobiomodulation is proposed as a non-linear process, and only low-level laser therapy (LLLT) is assumed to stimulate exposed cells, whereas high powered laser and fluences can cause negative effects, exhausting the cell's energy reserve as a consequence of excessive photon-based stimulation. In our work, we investigated and compared the effects of 808-nm diode laser (CW) with a new flat-top handpiece. To this purpose, we tested the photobiomodulation effects of 1 and 3 J/cm(2) fluence, both generated by 100 mW or 1 W of laser power and of 64 J/cm(2) of fluence generated by 100 mW, 1 W, 1.5 W or 2 W, as expressed through oxygen consumption and ATP synthesis of Paramecium. Data collected indicates the incremental consumption of oxygen through irradiation with 3 J/cm(2)-100 mW or 64 J/cm(2)-1 W correlates with an increase in Paramecium ATP synthesis. The Paramecium respiration was inhibited by fluences 64 J/cm(2)-100 mW or 64 J/cm(2)-2 W and was followed by a decrease in the endogenous ATP concentration. The 1 J/cm(2)-100 mW or 1 W and 3 J/cm(2)-1 W did not affect mitochondrial activity. The results show that the fluence of 64 J/cm(2)-1 W more than the 3 J/cm(2)-100 mW causes greater efficiency in Paramecium mitochondria respiratory chain activity. Our results suggest that thanks to flat-top handpiece we used, high fluences by high-powered laser have to be reconsidered as an effective and non-invasive therapy. Possible associated benefits of deeper tissue penetration would increase treatment effectiveness and reduced irradiation time.

In Paramecium, cilia beating is correlated to intracellular calcium concentration ([Ca2+ ]i) and nitric oxide (NO) synthesis. Recent findings affirm that photobiomodulation (PBM) can transiently increase the [Ca2+ ]i in mammalian cells. In this study, we investigated the effect of both 808 and 980 nm diode laser irradiated with flat-top hand-piece on [Ca2+ ]i and NO production of Paramecium primaurelia, to provide basic information for the development of new therapeutic approaches. In the experiments, the laser power in CW varied (0.1; 0.5; 1; and 1.5 W) to generate the following respective fluences: 6.4; 32; 64; and 96 J cm-2 . The 6.4 J cm-2 did not induce PBM if irradiated by both 808 and 980 nm diode laser. Conversely, the 32 J cm-2 fluence had no effect on Paramecium cells if irradiated by the 808 nm laser, while if irradiated by the 980 nm laser induced increment in swimming speed (suggesting an effect on the [Ca2+ ]i, NO production, similar to the 64 J cm-2 with the 808 nm wavelength). The more evident discordance occurred with the 96 J cm-2 fluence, which had the more efficient effect on PBM among the parameters if irradiated with the 808 nm laser and killed the Paramecium cells if irradiated by the 980 nm laser. Lastly, the 980 nm and 64 or 96 J cm-2 were the only parameters to induce a release of stored calcium.

Many investigations on protists indicate that they play an important role in agricultural soils. We have tested the effects of three organophosphate (OP) pesticides, basudin, cidial, and fenix, on the soil ciliate Colpoda inflata, and examined its viability, fission rate, ability to excyst and extrude macronuclear chromatin into cytoplasm. Exposure to these OPs caused a dose-dependent effect on cell viability, and significantly reduced the mean fission rate at a concentration of 1/10(5) v/v. After exposure of resting cysts to 1/10(5) v/v or 1/10(6) v/v concentrations of basudin or cidial, the number of excysted cells was significantly lower than that of the controls. Conversely, exposure to a 1/10(5) v/v fenix concentration did not affect excystment and exposure to 1/10(6) v/v was found to promote excystment. Moreover, exposure to these OPs (1/10(4) v/v or 1/10(5) v/v) interferred with the ability to extrude macronuclear chromatin. The median lethal concentration in 60 min for each OPs tested was at least a hundred times lower than the doses recommended by the manufacturer. Finally, as the inhibition of cholinesterase (ChE) activity is the first target of OPs, the presence of ChE activity was checked in C. inflata. Three ChE activities were found, hydrolyzing the substrates acetyl-beta-methyl thiocholine iodide, propionyl thiocholine iodide and butyryl thiocholine iodide, that appeared to be very low and not inhibited by OP-exposure.

It is now widely accepted that Protists are relevant bioassays to be exploited for the study of environmental modifications due to the presence of xenobiotic compounds. In this work, we evaluated the possibility of utilizing Euplotes crassus, an interstitial marine ciliate, for the pre-chemical screening of environmental sites, such as estuarine and coastal sediments. With this aim, we tested the sensitivity of E. crassus to exposure to three classes of pollutants: an organophosphate neurotoxic drug, basudin, largely used for pest control in agricultural sites, a toxic heavy metal, mercury (HgCl2), and an aromatic polycyclic hydrocarbon, benzopyrene (BP). We found a dose-dependent effect of these compounds on cell viability at concentrations ranging from 1/102 v/v to 1/107 v/v for basudin, from 5 μM to 0.1 μM for HgCl2, and from 50 μM to 1 μM for BP. In particular, 100% mortality was caused by a 1-h exposure to 1/105 v/v basudin, or 2 μM HgCl2, or 25 μM BP, and by a 24-h exposure to 1/106 v/v basudin, 0.5 μM HgCl2, or 5 μM BP. A significant decrease in the daily mean fission rate (P

Recently, we have shown the presence of molecules belonging to the cholinergic signalling system in the ciliate Paramecium primaurelia and in the sarcodine Dictyostelium discoideum. Propionylcholinesterase (PrChE) activity has been detected in single-cell amoebae of D. discoideum, using cytochemical, electrophoretic, and spectrophotometric methods (Falugi et al., Chemosphere, 48:407–414, 2002; Amaroli et al., Europ. J. Protistol., 39:213–222, 2003). It has been suggested that this enzyme activity is involved in cell–cell and cell–environment interactions, as its inhibition by xenobiotic compounds affects cell migration and aggregation. In this work, we have spectrophotometrically evaluated the effect of an ELF-EMF of 300 T, 50 Hz, on PrChE activity after exposure of single-cell amoebae from 1 h up to 48 h at 22°C. The enzyme activity was inhibited significantly by 1-h- and 3-h exposures, whereas it was similar to the control value after a 4-h exposure. A significant increase in PrChE activity was found after 5- and 24-h exposures, while a decrease in PrChE activity appeared in 48-h-exposed samples. The increased PrChE activity detected in 24-h-exposed cells returned to the control value 24 h after transferring the amoebae to standard conditions. A delay in both migration and aggregation processes was observed in 3 h-exposed cells, corresponding to a decreased PrChE activity. After a 24-h exposure, a decrease in the fission rate and an increase in the cell size were found.

Previous results of our study carried out on Dictyostelium discoideum showed the presence in single-cell amoebae of both cholinesterase (ChE) activity, able to hydrolyze the propionyl thiocholine iodide substrate, and “true” acetylcholinesterase (AChE) activity, similar to that of Electrophorus electricus . These activities are sensitive to anti-ChE agents, and to the inhibitors classically used to discriminate different ChEs. In this work, we have investigated the presence of ChE activities and of both acetylcholine (ACh) nicotinic and muscarinic receptors, during the developmental cycle of D. discoideum . The spectrophotometric evaluation showed that slugs displayed the highest enzyme activities, AChE being the main activity, while fruiting bodies exhibited the lowest. The presence of AChE activity was cytochemically detected in all the cell populations of the developmental cycle, except in the fruiting bodies where it was localized exclusively to the level of the spores. Molecules recognized by the anti-butyrylcholinesterase antibody were detected in single-cell amoebae, first fingers, slugs, and culminants, except in the fruiting bodies where they were found exclusively in the spore walls. The ACh nicotinic receptors were cytochemically identified in single-cell amoebae, slugs, and spores; however, molecules immunologically related to the ACh muscarinic receptors were not present in the spores.

Photobiomodulation (PBM) has been used in clinical practice for more than 40 years. To clarify the mechanisms of action of PBM at cellular and organism levels, we investigated its effect on Paramecium primaurelia (Protozoa) irradiated by an 808 nm infrared diode laser with a flat-top handpiece (1 W in CW). Our results led to the conclusion that: (1) the 808 nm laser stimulates the P. primaurelia without a thermal effect, (2) the laser effect is demonstrated by an increase in swimming speed and in food vacuole formation, (3) the laser treatment affects endogenous adenosine triphosphate (ATP) production in a positive way, (4) the effects of irradiation dose suggest an optimum exposure time of 50 s (64 J cm(-2) of fluence) to stimulate the Paramecium cells; irradiation of 25 s shows no effect or only mild effects and irradiation up to 100 s does not increase the effect observed with 50 s of treatment, (5) the increment of endogenous ATP concentration highlights the positive photobiomodulating effect of the 808 nm laser and the optimal irradiation conditions by the flat-top handpiece.

In Dictyostelium discoideum (D. discoideum), compounds generating nitric oxide (NO) inhibit its aggregation and differentiation without altering cyclic guanosine monophosphate (cGMP) production. They do it by preventing initiation of cyclic adenosine monophosphate (cAMP) pulses. Furthermore, these compounds stimulate adenosine diphosphate (ADP)-ribosylation of a 41 kDa cytosolic protein and regulate the glyceraldehyde-3-phospate dehydrogenase activity. Yet, although D. discoideum cells produce NO at a relatively constant rate at the onset of their developmental cycle, there is still no evidence of the presence of nitric oxide synthase (NOS) enzymes. In this work, we detect the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in D. discoideum and we characterise it by specific inhibitors and physical-chemical conditions that allegedly distinguish between NOS-related and -unrelated NADPH-d activity.

Nitric oxide is a regulative molecule with important roles in the olfactory system of vertebrates. Chondrichtyans have a key position in vertebrate evolution and nothing is known about nitric oxide in their olfactory system. Aim of this work was to investigate the neuronal nitric oxide synthase (nNOS) immunoreactivity in the olfactory system of the shark Scyliorhinus canicula. Because nitric oxide is often related to GABA in the olfactory system, also the distribution of GABA and its synthesis enzyme GAD has been investigated. In the olfactory epithelium scattered cells in the basal and medial zone of the epithelium thickness presented nNOS-like immunoreactivity. In the olfactory bulb the nNOS-like immunoreactivity has been highlighted in nerve fibers around some blood vessels and in scattered GABAergic granule cells. The presence of nNOS in the olfactory system of S. canicula is overall lesser than that described in other vertebrates, even if nitric oxide probably keeps some essential functions.

It is now widely accepted that assays with protists are relevant to be exploited for the study of environmental modifications due to the presence of xenobiotic compounds. In this work, the possibility of utilizing Euplotes crassus, an interstitial marine ciliate, for the pre-chemical screening of estuarine and coastal sediments was evaluated. For this purpose, the effects of exposure to pollutants were tested on the cell viability, fission rate and lysosomal membrane stability of E. crassus. The following toxicants were used: an organophosphate (OP) pesticide, basudin, an organochlorine hydrocarbon, AFD25, both employed especially for pest control in agricultural sites, a toxic heavy metal, mercury (HgCl2) and different mixtures of the above-mentioned compounds, as they might occur in polluted sites. Exposure to these toxicants affected cell viability at concentrations ranging from 96.6 to 966 x 10(3)mg/l for basudin, from 3.3 to 33 x 10(3)mg/l for AFD25 and from 0.1 to 1mg/l for HgCl2. A significant decrease in the mean fission rate (P0.001) was found after 24- or 48-h exposures to 9.66 mg/l basudin, 3.3 mg/l AFD25 and 7 x 10(-2)mg/l HgCl2. Furthermore, the Neutral Red Retention Assay showed a significant decrease in lysosomal membrane stability after 60- and 120-min exposures to AFD25 (33 mg/l) and HgCl2 (0.33 mg/l). In addition, as it is well-known that the inhibition of acetylcholinesterase activity represents a specific biomarker of exposure to OP and carbamate pesticides in higher organisms, initially the presence of cholinesterase (ChE) activity was detected in E. crassus, using cytochemical, spectrophotometric and electrophoretic methods. Afterwards, this enzyme activity was characterized spectrophotometrically by its sensitivity to specific ChE inhibitors and to variations in pH and temperature. The ChE activity was inhibited significantly by basudin- (9.66 and 96.6 mg/l) or AFD25-exposure (3.3 mg/l). Conversely, exposure to AFD25 (33 mg/l) or HgCl2 (0.1 and 0.3mg/l) caused a significant increase in this enzyme activity. Moreover, exposure to mixtures containing basudin, AFD25 and HgCl2 was found to affect the cell viability, the mean fission rate and the ChE activity differently, in an unpredictable manner. Our results indicate that E. crassus seems to be a suitable test organism to evaluate the toxicity of marine sediments.

Recently, we showed that Paramecium primaurelia synthesizes molecules functionally related to the cholinergic system and involved in modulating cell-cell interactions leading to the sexual process of conjugation. It is known that nitric oxide (NO) plays a role in regulating the release of transmitter molecules, such as acetylcholine, and that the NO biosynthetic enzyme, nitric oxide synthase (NOS), shows nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity. In this work, we detected the presence of NADPH-d activity in P. primaurelia. We characterized this activity histochemically by examining its specificity for beta-NADPH and alpha-NADH co-substrates, and sensitivity both to variations in chemico-physical parameters and to inhibitors of enzymes showing NADPH-d activity. Molecules immunologically related to NOS were recognized by the anti-rat brain NOS (bNOS) antibody. Moreover, bNOS immunoreactivity and NADPH-d activity sites were found to be co-localized. The non-denaturing electrophoresis, followed by exposure to beta-NADPH or alpha-NADH co-substrates, revealed the presence of a band of apparent molecular mass of about 124 kDa or a band of apparent molecular mass of about 175 kDa, respectively. In immunoblot experiments, the bNOS antibody recognized a single band of apparent molecular mass of about 123 kDa.

The presence and function of acetylcholinesterase (AChE) in blood cells and plasma is well known. The enzyme activity is associated to red blood cell membranes, as well as to the perinuclear region of leucocytes, suggesting a role in the function of the latter. Recently coelomocytes, acknowledged progenitors of immune cells in vertebrate systems, have been shown to respond to stress conditions, both in laboratory experiments and field studies. Bearing in mind the similarity between coelomocytes and vertebrate blood cells, we investigated the presence of AChE activity in coelomocytes of the sea urchin Paracentrotus lividus characterized by specific inhibitors, in relation to cold stress induced in the laboratory. Exposure to stress was followed by electrophoretic analysis of AChE activity and Western blot analysis of AChE-like immunoreactivity. Moreover, quantitative analysis of the different molecular forms of AChE was carried out with the aid of a spectrophotometric method. The effects of stress on AChE activity were time dependent. In fact, the enzyme activity was detected by the catalytic activity in cleaving the substrate acetyl thiocholine iodide, which appears to be formed by different isoforms of the enzyme, compared to that of Electrophorus, which was used as a control. Western blot analyses showed the presence of different immunoreactive molecules, with molecular mass ranging from 265 kDa (corresponding to Electrophorus molecules) to about 180 kDa. According to quantitative analysis, the isoforms that were differentiated corresponded to an AChE with the capacity to cleave acetyl-β-methyl thiocholine iodide (the so-called true AChE, E.C. 3.1.1.7, corresponding to the 265 kDa band) and to a ChE with the capacity to cleave propionyl thiocholine iodide as well. The latter is an ancestral form of AChE, mainly present in invertebrates. Each of these forms was affected differently by varying time exposures.

Recently, we found acetylcholinesterase (AChE) activity in the ciliate protozoan Paramecium primaurelia. As in the slime mould Dictyostelium discoideum the presence of a serine esterase was found with strong sequence identity to Torpedo AChE, we extended to D. discoideum the investigation on the characterization and possible functions of cholinesterases (ChEs). In amoeboid cells, histochemical, biochemical, and electrophoresis analyses evidenced both a ChE activity able to hydrolyze the substrate PrTChI, and AChE (E.C. 3.1.1.7.) activity similar to Electrophorus electricus AChE. Conversely, butyrylcholinesterase activity was nearly absent, according to our previous results on P. primaurelia. Moreover, the possibility to utilize D. discoideum in a bioassay for the pre-chemical screening both of moist environments and fresh waters, in relation to the occurrence of the neurotoxic organophosphate drugs, such as "basudin", inhibiting ChE activity, was investigated. Exposure to basudin inhibited propionylcholinesterase (PrChE) activity in a dose-dependent manner in the range 10(-1)-10(-7) M (60% at 10(-4) M), without any significant effect on AChE activity. PrChE activity was inhibited slightly by 10(-5) M eserine, and reduced significantly both by 10(-5) M iso-OMPA and BW284C51, classically used to discriminate the different ChE molecular forms. The effects on cell morphology, cell density, and differentiation were evaluated in cultures exposed to PrTChI 10(-5) M or basudin 10(-4) M for a three-day period. The PrTChI-exposed sample exhibited cell morphology, cell density, ability to aggregate, and to form fruiting bodies similar to the control; whereas, the basudin-exposed sample showed anomalies in cell morphology and lower cell density than the control, together with inability to aggregate.