On the thermal impact during drilling operations in guided dental surgery: An experimental and numerical investigation.

In recent years, a major development in dental implantology has been the introduction of patient-specific 3D-printed surgical guides. The utilization of dental guides offers advantages such as enhanced accuracy in locating the implant sites, greater simplicity, and reliability in performing bone drilling operations. However, it is important to note that the presence of such guides may contribute to a rise in cutting temperature, hence increasing the potential hazards of thermal injury to the patient's bone. The aim of this study is to examine the drilling temperature evolution in two distinct methods for 3D-printed surgical dental guides, one utilizing an internal metal bushing system and the other using external metal reducers. Cutting tests are done on synthetic polyurethane bone jaw models using a lab-scale automated Computer Numeric Control (CNC) machine to find out the temperature reached by different drilling techniques and compare them to traditional free cutting configurations. Thermal imaging and thermocouples, as well as the development of numerical simulations using finite element modeling, are used for the aim. The temperature of the tools' shanks experienced an average rise of 2.4 °C and 4.8 °C, but the tooltips exhibited an average increase of around 17 °C and 24 °C during traditional and guided dental surgery, respectively. This finding provides confirmation that both guided technologies have the capability to maintain temperatures below the critical limit for potential harm to bone and tissue. Numerical models were employed to validate and corroborate the findings, which exhibited identical outcomes when applied to genuine bone samples with distinct thermal characteristics.

Insights into Cadmium-Induced Carcinogenesis through an In Vitro Study Using C3H10T1/2Cl8 Cells: The Multifaceted Role of Mitochondria.

In this paper, we report the metabolic characterization of two foci, F1 and F3, obtained at the end of Cell Transformation Assay (CTA), performed by treating C3H10T1/2Cl8 mouse embryo fibroblasts with 1 µM CdCl2 for 24 h. The elucidation of the cadmium action mechanism can be useful both to improve the in vitro CTA and to yield insights into carcinogenesis. The metabolism of the two foci was investigated through Seahorse and enzyme activity assays; mitochondria were studied in confocal microscopy and reactive oxygen species were detected by flow cytometry. The results showed that F1 focus has higher glycolytic and TCA fluxes compared to F3 focus, and a more negative mitochondrial membrane potential, so that most ATP synthesis is performed through oxidative phosphorylation. Confocal microscopy showed mitochondria crowded in the perinuclear region. On the other hand, F3 focus showed lower metabolic rates, with ATP mainly produced by glycolysis and damaged mitochondria. Overall, our results showed that cadmium treatment induced lasting metabolic alterations in both foci. Triggered by the loss of the Pasteur effect in F1 focus and by mitochondrial impairment in F3 focus, these alterations lead to a loss of coordination among glycolysis, TCA and oxidative phosphorylation, which leads to malignant transformation.

Combined Effects of Pesticides and Electromagnetic-Fields on Honeybees: Multi-Stress Exposure.

Honeybee and general pollinator decline is extensively reported in many countries, adding new concern to the general biodiversity loss. Many studies were addressed to assess the causes of pollinator decline, concluding that in most cases multi-stress effects were the most probable ones. In this research, the combined effects of two possible stress sources for bees, pesticides and electromagnetic fields (multi-stress conditions), were analyzed in the field. Three experimental sites were chosen: a control one far from direct anthropogenic stress sources, a pesticide-stress site and multi-stress one, adding to the same exposure to pesticides the presence of an electromagnetic field, coming from a high-voltage electric line. Experimental apiaries were monitored weekly for one year (from April 2017 to April 2018) by means of colony survival, queen activity, storage and brood amount, parasites and pathogens, and several biomarkers in young workers and pupae. Both exposure and effect biomarkers were analysed: among the first, acetylcholinesterase (AChE), catalase (CAT), glutathione S-transferase (GST) and alkaline phosphatase (ALP) and Reactive Oxygen Species (ROS); and among the last, DNA fragmentation (DNAFRAGM) and lipid peroxidation (LPO). Results showed that bee health conditions were the worst in the multi-stress site with only one colony alive out of the four ones present at the beginning. In this site, a complex picture of adverse effects was observed, such as disease appearance (American foulbrood), higher mortality in the underbaskets (common to pesticide-stress site), behavioral alterations (queen changes, excess of honey storage) and biochemical anomalies (higher ALP activity at the end of the season). The overall results clearly indicate that the multi-stress conditions were able to induce biochemical, physiological and behavioral alterations which severely threatened bee colony survival.

Manufacturing Signatures of Injection Molding and Injection Compression Molding for Micro-Structured Polymer Fresnel Lens Production.

Injection compression molding (ICM) provides enhanced optical performances of molded polymer optics in terms of birefringence and transmission of light compared to Injection molding (IM). Nevertheless, ICM requires case-dedicated process optimization to ensure that the required high accuracy geometrical replication is achieved, particularly especially in the case of surface micro-features. In this study, two factorial designs of experiments (DOE) were carried out to investigate the replication capability of IM and ICM on a micro structured Fresnel lens. A laser scanning confocal microscope was employed for the quality control of the optical components. Thus, a detailed uncertainty budget was established for the dimensional measurements of the replicated Fresnel lenses, considering specifically peak-to-valley (PV) step height and the pitch of the grooves. Additional monitoring of injection pressure allowed for the definition of a manufacturing signature, namely, the process fingerprint for the evaluation of the replication fidelity under different process conditions. Moreover, considerations on the warpage of parts were related to a manufacturing signature of the molding processes. At last, the global part mass average and standard deviation were measured to correlate local geometrical replication performances with global part quality trends.

Probing the Influence of Linker Length and Flexibility in the Design and Synthesis of New Trehalase Inhibitors.

This work aims to synthesize new trehalase inhibitors selective towards the insect trehalase versus the porcine trehalase, in view of their application as potentially non-toxic insecticides and fungicides. The synthesis of a new pseudodisaccharide mimetic 8, by means of a stereoselective α-glucosylation of the key pyrrolizidine intermediate 13, was accomplished. The activity of compound 8 as trehalase inhibitor towards C.riparius trehalase was evaluated and the results showed that 8 was active in the µM range and showed a good selectivity towards the insect trehalase. To reduce the overall number of synthetic steps, simpler and more flexible disaccharide mimetics 9-11 bearing a pyrrolidine nucleus instead of the pyrrolizidine core were synthesized. The biological data showed the key role of the linker chain's length in inducing inhibitory properties, since only compounds 9 (α,ß-mixture), bearing a two-carbon atom linker chain, maintained activity as trehalase inhibitors. A proper change in the glucosyl donor-protecting groups allowed the stereoselective synthesis of the ß-glucoside 9ß, which was active in the low micromolar range (IC50 = 0.78 µM) and 12-fold more potent (and more selective) than 9α towards the insect trehalase.

Natural variability of biochemical biomarkers in the macro-zoobenthos: Dependence on life stage and environmental factors.

Biomarkers are widely used in ecotoxicology as indicators of exposure to toxicants. However, their ability to provide ecologically relevant information remains controversial. One of the major problems is understanding whether the measured responses are determined by stress factors or lie within the natural variability range. In a previous work, the natural variability of enzymatic levels in invertebrates sampled in pristine rivers was proven to be relevant across both space and time. In the present study, the experimental design was improved by considering different life stages of the selected taxa and by measuring more environmental parameters. The experimental design considered sampling sites in 2 different rivers, 8 sampling dates covering the whole seasonal cycle, 4 species from 3 different taxonomic groups (Plecoptera, Perla grandis; Ephemeroptera, Baetis alpinus and Epeorus alpicula; Tricoptera, Hydropsyche pellucidula), different life stages for each species, and 4 enzymes (acetylcholinesterase, glutathione S-transferase, alkaline phosphatase, and catalase). Biomarker levels were related to environmental (physicochemical) parameters to verify any kind of dependence. Data were statistically elaborated using hierarchical multilevel Bayesian models. Natural variability was found to be relevant across both space and time. The results of the present study proved that care should be paid when interpreting biomarker results. Further research is needed to better understand the dependence of the natural variability on environmental parameters. Environ Toxicol Chem 2017;36:3158-3167. © 2017 SETAC.

Natural variability of enzymatic biomarkers in freshwater invertebrates.

Biomarkers have been widely employed in ecotoxicology as early warning indicators of exposure to toxicants. Very often, they are used to compare reference and polluted sites, or to analyse time trends. However, very few studies focus on the natural variability range of biomarkers in the environment, which is pivotal to understand if the detected differences are actually determined by any adverse effects due to pollution. This work assesses the natural spatio-temporal variability of some enzymatic levels, frequently used as biomarkers, in freshwater benthic invertebrates. The influence of some environmental parameters on the enzymatic levels was also evaluated. Three families of insect larvae (Perlidae, Baetidae, and Heptageniidae) were sampled in three pristine streams and in eight different dates. Four enzymes (acetylcholinesterase, glutathione-S-transferase, alkaline phosphatase, and catalase) were measured. The natural variability of enzymatic levels was often significant in all considered species across both space and time. The observed pattern was poorly explained by the monitored environmental parameters. The results of this work show that great care should be paid when interpreting monitoring data in which biomarker levels are measured and compared among sites or dates. Presuming that measured differences are due to anthropogenic factors can be misleading, when other potentially influencing factors have not been accounted for.

Physico-chemical properties and biological effects of diesel and biomass particles.

Diesel combustion and solid biomass burning are the major sources of ultrafine particles (UFP) in urbanized areas. Cardiovascular and pulmonary diseases, including lung cancer, are possible outcomes of combustion particles exposure, but differences in particles properties seem to influence their biological effects. Here the physico-chemical properties and biological effects of diesel and biomass particles, produced under controlled laboratory conditions, have been characterized. Diesel UFP were sampled from a Euro 4 light duty vehicle without DPF fuelled by commercial diesel and run over a chassis dyno. Biomass UFP were collected from a modern automatic 25 kW boiler propelled by prime quality spruce pellet. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of both diesel and biomass samples showed aggregates of soot particles, but in biomass samples ash particles were also present. Chemical characterization showed that metals and PAHs total content was higher in diesel samples compared to biomass ones. Human bronchial epithelial (HBEC3) cells were exposed to particles for up to 2 weeks. Changes in the expression of genes involved in xenobiotic metabolism were observed after exposure to both UFP already after 24 h. However, only diesel particles modulated the expression of genes involved in inflammation, oxidative stress and epithelial-to-mesenchymal transition (EMT), increased the release of inflammatory mediators and caused phenotypical alterations, mostly after two weeks of exposure. These results show that diesel UFP affected cellular processes involved in lung and cardiovascular diseases and cancer. Biomass particles exerted low biological activity compared to diesel UFP. This evidence emphasizes that the study of different emission sources contribution to ambient PM toxicity may have a fundamental role in the development of more effective strategies for air quality improvement.

A new method and tool for detection and quantification of PM oxidative potential.

Airborne particulate matter (PM) contains several quinones, which are able to generate reactive oxygen species impacting on cell viability. A method able to detect and quantify PM oxidative potential, based on the cytochrome c (cyt-c) reduction by means of superoxide anion produced through quinones redox cycling in the presence of reducing agents, is here described. Tris(2-carboxyethyl)phosphine resulted to be the most efficient reducing agent among the ones tested. The procedure included rapid particles extraction, followed by two alternative analytical methods, a spectrophotometric assay based on the initial rate of cyt-c reduction at 550 nm, and an amperometric assay, based on self-assembled monolayers modified gold electrodes. The smallest amount of PM needed to obtain an evaluable signal is 2 µg. The described procedure may represent a starting point to develop devices for PM measurements in polluted atmospheric environments.

New synthesis and biological evaluation of uniflorine A derivatives: towards specific insect trehalase inhibitors.

7-Deoxy-uniflorine A (6), synthesized ex novo with a straightforward and simple strategy, and the analogues 4, 5 and 7, were evaluated as potential inhibitors of insect trehalase from Chironomus riparius and Spodoptera littoralis. All the compounds were tested against porcine trehalase as the mammalian counterpart and α-amylase from human saliva as a relevant glucolytic enzyme. The aim of this work is the identification of the simplest pyrrolizidine structure necessary to impart selective insect trehalase inhibition, in order to identify new specific inhibitors that can be easily synthesized compared to our previous reports with the potential to act as non-toxic insecticides and/or fungicides. All the derivatives 4­7 proved to be active (from low micromolar to high nanomolar range activity) towards insect trehalases, while no activity was observed against α-amylase. In particular, the natural compound uniflorine A and its 7-deoxy analogue were found to selectively inhibit insect trehalases, as they are inactive towards the mammalian enzyme. The effect of compound 6 was also analyzed in preliminary in vivo experiments. These new findings allow the identification of natural uniflorine A and its 7-deoxy analogue as the most promising inhibitors among a series of pyrrolizidine derivatives for future development in the agrochemical field, and the investigation also outlined the importance of the stereochemistry at C-6 of pyrrolizidine nucleus to confer such enzyme specificity.

N-Bridged 1-deoxynojirimycin dimers as selective insect trehalase inhibitors.

A small set of N-bridged 1-deoxynojirimycin dimers has been synthesized and evaluated as potential inhibitors of insect trehalase from midge larvae of Chironomus riparius, porcine trehalase as the mammalian counterpart and α-amylase from human saliva. All the tested compounds (2-4) proved to be active (micromolar range activity) against insect trehalase, showing selectivity toward the insect glycosidase. No activity was observed against α-amylase.

Molecular cloning of soluble trehalase from Chironomus riparius larvae, its heterologous expression in Escherichia coli and bioinformatic analysis.

Trehalase is involved in the control of trehalose concentration, the main blood sugar in insects. Here, we describe the molecular cloning of the cDNA encoding for the soluble form of the trehalase from the midge larvae of Chironomus riparius, a well-known bioindicator of the quality of freshwater environments. Molecular cloning was achieved through multiple alignment of Diptera trehalase sequences, allowing the synthesis of internal homology-based primers; the complete open reading frame(ORF) was subsequently obtained through RACE-PCR(where RACE is rapid amplification of cDNA ends). The cDNA contained the 5' untranslated region (UTR), the 3' UTR including a poly(A) tail and the ORF of 1,725 bp consisting of 574 amino acid residues with a predicted molecular mass of 65,778 Da. Recombinant trehalase was successfully expressed in Escherichia coli as a His-tagged protein and purified on Ni-NTA affinity chromatography. Primary structure analysis showed a series of characteristic features shared by all insect trehalases, while three-dimensional structure prediction yielded the typical glucosidase fold, the two key residues involved in the catalytic mechanism being conserved. Production of recombinant insect trehalases opens the way to structural characterizations of the catalytic site, which might represent, among others, an element for reconsidering the enzyme as a target in pest insects' control.

Synthesis and biological evaluation of nojirimycin- and pyrrolidine-based trehalase inhibitors.

A small set of nojirimycin- and pyrrolidine-based iminosugar derivatives has been synthesized and evaluated as potential inhibitors of porcine and insect trehalases. Compounds 12, 13 and 20 proved to be active against both insect and porcine trehalases with selectivity towards the insect glycosidase, while compounds 10, 14 and 16 behaved as inhibitors only of insect trehalase. Despite the fact that the activity was found in the micromolar range, these findings may help in elucidating the structural features of this class of enzymes of different origin, which are still scarcely characterised.

A membrane-bound trehalase from Chironomus riparius larvae: purification and sensitivity to inhibition.

A preparation of a membrane-bound trehalase from the larvae of the midge Chironomus riparius (Diptera: Chironomidae) was obtained by detergent solubilization, ion-exchange chromatography and concanavalin A affinity chromatography. Trehalase was purified 1080-fold to a specific activity of 75 U mg(-)(1). The initial rate of trehalase activity followed Henri-Michaelis-Menten kinetics with a K(m) of 0.48 +/- 0.04 mM. Catalytic efficiency was maximal at pH 6.5. The activity was highly inhibited by mono- and bicyclic iminosugar alkaloids such as (in order of potency) casuarine (IC(50) = 0.25 +/- 0.03 microM), deoxynojirimycin (IC(50) = 2.83 +/- 0.34 microM) and castanospermine (IC(50) = 12.7 +/- 1.4 microM). Increasing substrate concentration reduced the inhibition. However, in the presence of deoxynojirimycin, Lineweaver-Burk plots were curvilinear upward. Linear plots were obtained with porcine trehalase. Here, we propose that deoxynojirimycin inhibits the activity of trehalase from C. riparius according to a ligand exclusion model. Inhibition was further characterized by measuring enzyme activity in the presence of a series of casuarine and deoxynojirimycin derivatives. For comparison, inhibition studies were also performed with porcine trehalase. Results indicate substantial differences between midge trehalase and mammalian trehalase suggesting that, in principle, inhibitors against insect pests having trehalase as biochemical targets can be developed.

Casuarine-6-O-alpha-D-glucoside and its analogues are tight binding inhibitors of insect and bacterial trehalases.

Two novel casuarine-6-alpha-D-glucoside analogues, as well as the parent compound, were synthesized and tested as inhibitors towards Chironomus riparius, mammalian pig kidney and Escherichia coli trehalases. Their potent and selective activity is promising for the development of new insecticides.

Effects of river pollution on the colonisation of artificial substrates by macrozoobenthos.

The impact of pesticides on macrobenthos communities was studied on two aquatic environments in Northern Italy; River Meolo, a site exposed to agricultural pollution, and River Upper Livenza a low pollution reference site. Colonisation of artificial substrates was compared throughout the productive season. The relevance of multiple environmental stressors other than pesticides (e.g. oxygen depletion) was also assessed. Biochemical indicators (enzyme and metabolite biomarkers) were measured on selected organisms. Biomarker results (acetylcholinesterase, glutathione S-transferase, and napthylacetate esterase), as well as community structure patterns, revealed a significant difference between the two rivers. Cause-effect relationships between results and multiple stress factors were discussed.

Antioxidant defenses preserve membrane transport activity in Chironomus riparius larvae exposed to anoxia.

Changes in enzyme activities, metabolite concentrations, and membrane transport activity underlying the Chironomus riparius larvae adaptive response to anoxia were investigated. Trehalose, malate, and aspartate degradation and alanine accumulation were recorded. During anoxia exposure, there was a boost of antioxidant defenses as shown by an increase of the specific activity of the enzymes catalase, glutathione-S-transferase, glutathione peroxidase, glutathione-synthase, malic enzyme, and NADP-dependent isocitrate dehydrogenase. The ratio, glutathione reduced over glutathione oxidized, decreased. Except for alanine and catalase, the parameters return to their basal value when larvae are transferred to normoxic conditions. To test whether antioxidant defenses had protective effects on membrane functionality, L-leucine uptake into brush border membrane vesicles and membrane lipid peroxidation was measured. No difference between membranes prepared from larvae exposed to anoxia and control larvae was found. The amino acid alanine, when present inside the vesicles, trans-stimulated leucine uptake. This effect could represent a mechanism to stimulate amino acid uptake and catabolism in vivo when free alanine concentration increases during hypoxic periods.

Increased alanine concentration is associated with exposure to fenitrothion but not carbamates in Chironomus riparius larvae.

Chironomus riparius Meigen were exposed to three different insecticides, the organophosphorous fenitrothion and the carbamates carbaryl and carbofuran (0, 1, 10, and 100 microg/L) for 24h as fourth-instar larvae. Acetylcholinesterase (AChE), naphtylacetate esterase (NAE), p-nitrophenylacetate esterase (PNPAE), glutathione-S-transferase (GST), and a number of metabolites (alanine, pyruvate, lactate, trehalose, aspartate, oxalacetate) were measured to determine which was the most valuable biochemical biomarker of exposure. AChE activity was significantly reduced by all three insecticides, PNPAE by fenitrothion, carbofuran and carbaryl, whereas NAE activity was stimulated by carbaryl and unaffected by fenitrothion and carbofuran. Metabolites analysis revealed a strong accumulation of alanine in larvae exposed to fenitrothion, but not in larvae exposed to carbamates. This accumulation was accompanied by a significant increase of lactate and a significant decrease of pyruvate and trehalose. No variations were observed with carbofuran and carbaryl. No change of aspartate concentration was detected. We conclude that the association of alanine accumulation with a significant inhibition of AChE activity can be used as a valuable biochemical biomarker of exposure.

Leucine transport in brush border membrane vesicles from freshwater insect larvae.

Leucine transport across brush border membrane vesicles prepared from four insect species common to European freshwater streams has been characterized. The species studied were: Ephemera danica (Ephemeroptera: Ephemeridae), Isoperla grammatica (Plecoptera: Perlodidae), Hydropsyche pellucidula (Trichoptera: Hydropsychidae), and Hybomitra bimaculata (Diptera: Tabanidae). The transport differed among the studied taxa for several features, including pH and sodium dependence, substrate affinity and specificity, and efficiency. In H. pellucidula and E. danica, leucine uptake was higher at pH 7.4 than at more alkaline or acidic pH values, whereas in I. grammatica and H. bimaculata, the uptake was rather constant when pH varied from 5.0 to 7.4, then strongly decreased at pH 8.8. All but E. danica displayed a transient intravescicular leucine accumulation in the presence of sodium, suggesting the existence of a cation-leucine symport mechanism. The sodium dependence ranged according to the following order: H. pellucidula > I. grammatica > H. bimaculata > E. danica. Moreover, in H. pellucidula and I. grammatica, the sodium-dependence was stronger at pH 8.8 than at pH 7.4. In E. danica, leucine uptake was sodium-independent at all pH values. The highest value of V(max) (45.3 pmol.s(-1).mg proteins(-1)) was in E. danica, which, however, displayed the lowest affinity (K(m) 137 muM) when compared to the kinetic parameters of other taxa. The V(max) and K(m) values were: 40 and 52.5, 32.1 and 12.5, and 4.5 and 230 for H. bimaculata, H. pellucidula, and I. grammatica, respectively. The obtained results are discussed within our current knowledge of amino acid transport systems in insects.

Changes in leucine transport activity in Chironomus riparius larvae after short-term exposure to potassium dichromate and fenitrothion.

The effect of sublethal concentrations of potassium dichromate and fenitrothion on sodium-leucine cotransport in brush border membrane vesicles from Chironomus riparius larvae has been investigated. Exposure to potassium dichromate and fenitrothion caused a dose- and time-dependent inhibition of leucine uptake. Transport inhibition is easily detectable at doses 100-fold lower than LD50. Kinetic experiments showed that inhibition was mainly caused by a decrease of the Vmax (680 +/- 53 vs. 382 +/- 23 and 555 +/- 27 nmol/15s/mg protein in control and exposed larvae to K2Cr2O7 and fenitrothion, respectively). Inhibition is possibly related to a variation of sodium ions permeability as evidenced by increased membrane lipid peroxidation. Appropriate control experiments ruled out that the observed differences could be due to changes in general features of membrane preparations. Transport inhibition observed in larvae exposed to potassium dichromate was accompanied by changes in ascorbate peroxidase and dehydroascorbate reductase activities, whereas those exposed to fenitrothion displayed an increase in transaminase activity. The possible value of leucine uptake as biochemical biomarker is briefly discussed. Arch. Insect Biochem. Physiol. 55:90-101, 2004.