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Poly- and perfluoroalkyl substances (PFAS) are frequently detected in the environment and are linked to adverse reproductive health outcomes in humans. Although legacy PFAS have been phased out due to their toxicity, alternative PFAS are increasingly used despite the fact that information on their toxic effects on reproductive traits is particularly scarce. Here, we exposed male guppies (Poecilia reticulata) for a short period (21 days) to an environmentally realistic concentration (1 ppb) of PFOA, a legacy PFAS, and its replacement compound, GenX, to assess their impact on reproductive traits and gene expression. Exposure to PFAS did not impair survival but instead caused sublethal effects. Overall, PFAS exposure caused changes in male sexual behaviour and had detrimental effects on sperm motility. Sublethal variations were also seen at the transcriptional level, with the modulation of genes involved in immune regulation, spermatogenesis, and oxidative stress. We also observed bioaccumulation of PFAS, which was higher for PFOA than for GenX. Our results offer a comprehensive comparison of these two PFAS and shed light on the toxicity of a newly emerging alternative to legacy PFAS. It is therefore evident that even at low concentrations and with short exposure, PFAS can have subtle yet significant effects on behaviour, fertility, and immunity. These findings underscore the potential ramifications of pollution under natural conditions and their impact on fish populations.

Glioblastomas exhibit remarkable heterogeneity at various levels, including motility modes and mechanoproperties that contribute to tumor resistance and recurrence. In a recent study using gridded micropatterns mimicking the brain vasculature, glioblastoma cell motility modes, mechanical properties, formin content, and substrate chemistry are linked. Now is presented, SP2G (SPheroid SPreading on Grids), an analytic platform designed to identify the migratory modes of patient-derived glioblastoma cells and rapidly pinpoint the most invasive sub-populations. Tumorspheres are imaged as they spread on gridded micropatterns and analyzed by this semi-automated, open-source, Fiji macro suite that characterizes migration modes accurately. SP2G can reveal intra-patient motility heterogeneity with molecular correlations to specific integrins and EMT markers. This system presents a versatile and potentially pan-cancer workflow to detect diverse invasive tumor sub-populations in patient-derived specimens and offers a valuable tool for therapeutic evaluations at the individual patient level., (© 2024 The Authors. Small Methods published by Wiley‐VCH GmbH.)

Cytochrome P450 3A (CYP3A) enzymes catalyze the metabolism of a wide range of endogenous and exogenous compounds. Genetic variations in the 3 CYP3A isoforms (CYP3A28, CYP3A74, and CYP3A76) may influence their expression and activity, leading to inter-individual differences in xenobiotic metabolism. In domestic cattle, understanding how genetic variations modulate CYP3A activity is crucial for both its therapeutic implications (clinical efficacy and adverse drug effects) and food safety (residues in foodstuff). Here, we updated the variant calling of CYP3As in 300 previously sequenced Piedmontese beef cattle, using the most recent reference genome, which contains an updated, longer sequence for CYP3A28 . All but one previously identified missense variants were confirmed and a new variant, R105W in CYP3A28, was discovered. Through computational mutagenesis and molecular docking, we computationally predicted the impact of all identified CYP3A variant enzymes on protein stability and their affinity for aflatoxin B1 (AFB1), a potent carcinogen and food contaminant. For CYP3A28, we also computationally predicted its affinity for the probe substrate nifedipine (NIF). We found that CYP3A28 with R105W variant cannot accommodate NIF nor AFB1 in the binding pocket, thus affecting their metabolism. Our work provides computational foundation and prioritized ranking of CYP3A variants for future experimental validations.

Perfluoroalkyl substances (PFAS) are common environmental pollutants, but their toxicity framework remains elusive. This research focused on ten PFAS, evaluating their impacts on two ecotoxicologically relevant model organisms from distinct trophic levels: the crustacean Daphnia magna and the unicellular green alga Raphidocelis subcapitata. The results showed a greater sensitivity of R. subcapitata compared to D. magna. However, a 10-day follow-up to the 48 h immobilisation test in D. magna showed delayed mortality, underlining the limitations of relying on EC 50 s from standard acute toxicity tests. Among the compounds scrutinized, Perfluorodecanoic acid (PFDA) was the most toxic to R. subcapitata, succeeded by Perfluorooctane sulfonate (PFOS), Perfluorobutanoic acid (PFBA), and Perfluorononanoic acid (PFNA), with the latter being the only one to show an algicidal effect. In the same species, assessment of binary mixtures of the compounds that demonstrated high toxicity in the single evaluation revealed either additive or antagonistic interactions. Remarkably, with an EC 50 of 31 mg L -1 , the short-chain compound PFBA, tested individually, exhibited toxicity levels akin to the notorious long-chain PFOS, and its harm to freshwater ecosystems cannot be ruled out. Despite mounting toxicological evidence and escalating environmental concentrations, PFBA has received little scientific attention and regulatory stewardship. It is strongly advisable that regulators re-evaluate its use to mitigate potential risks to the environmental and human health., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Seafood is increasingly traded worldwide, but its supply chain is particularly prone to frauds. To increase consumer confidence, prevent illegal trade, and provide independent validation for eco-labelling, accurate tools for seafood traceability are needed. Here we show that the use of microbiome profiling (MP) coupled with machine learning (ML) allows precise tracing the origin of Manila clams harvested in areas separated by small geographic distances. The study was designed to represent a real-world scenario. Clams were collected in different seasons across the most important production area in Europe (lagoons along the northern Adriatic coast) to cover the known seasonal variation in microbiome composition for the species. DNA extracted from samples underwent the same depuration process as commercial products (i.e. at least 12 h in open flow systems)., Results: Machine learning-based analysis of microbiome profiles was carried out using two completely independent sets of data (collected at the same locations but in different years), one for training the algorithm, and the other for testing its accuracy and assessing the temporal stability signal. Briefly, gills (GI) and digestive gland (DG) of clams were collected in summer and winter over two different years (i.e. from 2018 to 2020) in one banned area and four farming sites. 16S DNA metabarcoding was performed on clam tissues and the obtained amplicon sequence variants (ASVs) table was used as input for ML MP. The best-predicting performances were obtained using the combined information of GI and DG (consensus analysis), showing a Cohen K-score > 0.95 when the target was the classification of samples collected from the banned area and those harvested at farming sites. Classification of the four different farming areas showed slightly lower accuracy with a 0.76 score., Conclusions: We show here that MP coupled with ML is an effective tool to trace the origin of shellfish products. The tool is extremely robust against seasonal and inter-annual variability, as well as product depuration, and is ready for implementation in routine assessment to prevent the trade of illegally harvested or mislabeled shellfish., (© 2024. The Author(s).)

Aflatoxin B1 (AFB1) is a pro-carcinogenic compound bioactivated in the liver by cytochromes P450 (CYPs). In mammals, CYP1A and CYP3A are responsible for AFB1 metabolism, with the formation of the genotoxic carcinogens AFB1-8,9-epoxide and AFM1, and the detoxified metabolite AFQ1. Due to climate change, AFB1 cereals contamination arose in Europe. Thus, cattle, as other farm animals fed with grains (pig, sheep and broiler), are more likely exposed to AFB1 via feed with consequent release of AFM1 in milk, posing a great concern to human health. However, knowledge about bovine CYPs involved in AFB1 metabolism is still scanty. Therefore, CYP1A1- and CYP3A74-mediated molecular mechanisms of AFB1 hepatotoxicity were here dissected. Molecular docking of AFB1 into CYP1A1 model suggested AFB1 8,9-endo- and 8,9-exo-epoxide, and AFM1 formation, while docking of AFB1 into CYP3A74 pointed to AFB1 8,9-exo-epoxide and AFQ1 synthesis. To biologically confirm these predictions, CYP1A1 and CYP3A74 knockout (KO) BFH12 cell lines were exposed to AFB1. LC-MS/MS investigations showed the abolished production of AFM1 in CYP1A1 KO cells and the strong increase of parent AFB1 in CYP3A74 KO cells; the latter result, coupled to a decreased cytotoxicity, suggested the major role of CYP3A74 in AFB1 8,9-exo-epoxide formation. Finally, RNA-sequencing analysis indirectly proved lower AFB1-induced cytotoxic effects in engineered cells versus naïve ones. Overall, this study broadens the knowledge on AFB1 metabolism and hepatotoxicity in cattle, and it provides the weight of evidence that CYP1A1 and CYP3A74 inhibition might be exploited to reduce AFM1 and AFBO synthesis, AFB1 toxicity, and AFM1 milk excretion., (© 2024. The Author(s).)

In human, the cytochrome P450 3A (CYP3A) subfamily of drug-metabolizing enzymes (DMEs) is responsible for a significant number of phase I reactions, with the CYP3A4 isoform superintending the hepatic and intestinal metabolism of diverse endobiotic and xenobiotic compounds. The CYP3A4-dependent bioactivation of chemicals may result in hepatotoxicity and trigger carcinogenesis. In cattle, four CYP3A genes (CYP3A74, CYP3A76, CYP3A28 and CYP3A24) have been identified. Despite cattle being daily exposed to xenobiotics (e.g., mycotoxins, food additives, drugs and pesticides), the existing knowledge about the contribution of CYP3A in bovine hepatic metabolism is still incomplete. Nowadays, CRISPR/Cas9 mediated knockout (KO) is a valuable method to generate in vivo and in vitro models for studying the metabolism of xenobiotics. In the present study, we successfully performed CRISPR/Cas9-mediated KO of bovine CYP3A74, human CYP3A4-like, in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP3A74 ablation was confirmed at the DNA, mRNA, and protein level. The subsequent characterization of the CYP3A74 KO clone highlighted significant transcriptomic changes (RNA-sequencing) associated with the regulation of cell cycle and proliferation, immune and inflammatory response, as well as metabolic processes. Overall, this study successfully developed a new CYP3A74 KO in vitro model by using CRISPR/Cas9 technology, which represents a novel resource for xenobiotic metabolism studies in cattle. Furthermore, the transcriptomic analysis suggests a key role of CYP3A74 in bovine hepatocyte cell cycle regulation and metabolic homeostasis., Competing Interests: Declaration of competing interest 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., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The cytochrome P450 1A (CYP1A) subfamily of xenobiotic metabolizing enzymes (XMEs) consists of two different isoforms, namely CYP1A1 and CYP1A2, which are highly conserved among species. These two isoenzymes are involved in the biotransformation of many endogenous compounds as well as in the bioactivation of several xenobiotics into carcinogenic derivatives, thereby increasing the risk of tumour development. Cattle (Bos taurus) are one of the most important food-producing animal species, being a significant source of nutrition worldwide. Despite daily exposure to xenobiotics, data on the contribution of CYP1A to bovine hepatic metabolism are still scarce. The CRISPR/Cas9-mediated knockout (KO) is a useful method for generating in vivo and in vitro models for studying xenobiotic biotransformations. In this study, we applied the ribonucleoprotein (RNP)-complex approach to successfully obtain the KO of CYP1A1 in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP1A1 excision was confirmed at the DNA, mRNA and protein level. Therefore, RNA-seq analysis revealed significant transcriptomic changes associated with cell cycle regulation, proliferation, and detoxification processes as well as on iron, lipid and mitochondrial homeostasis. Altogether, this study successfully generates a new bovine CYP1A1 KO in vitro model, representing a valuable resource for xenobiotic metabolism studies in this important farm animal species., (© 2024. The Author(s).)

Background: The reuse of dredged sediments in ports and lagoons is a big issue as it should not affect the quality and the equilibrium of ecosystems. In the lagoon of Venice, sediment management is of crucial importance as sediments are often utilized to built-up structures necessary to limit erosion. However, the impact of sediment reuse on organisms inhabiting this delicate area is poorly known. The Manila clam is a filter-feeding species of high economic and ecological value for the Venice lagoon experiencing a drastic decline in the last decades. In order to define the molecular mechanisms behind sediment toxicity, we exposed clams to sediments sampled from different sites within one of the Venice lagoon navigable canals close to the industrial area. Moreover, we investigated the impacts of dredged sediments on clam's microbial communities., Results: Concentrations of the trace elements and organic chemicals showed increasing concentrations from the city of Venice to sites close to the industrial area of Porto Marghera, where PCDD/Fs and PCBs concentrations were up to 120 times higher than the southern lagoon. While bioaccumulation of organic contaminants of industrial origin reflected sediments' chemical concentrations, metal bioaccumulation was not consistent with metal concentrations measured in sediments probably due to the activation of ABC transporters. At the transcriptional level, we found a persistent activation of the mTORC1 signalling pathway, which is central in the coordination of cellular responses to chemical stress. Microbiota characterization showed the over-representation of potential opportunistic pathogens following exposure to the most contaminated sediments, leading to host immune response activation. Despite the limited acquisition of new microbial species from sediments, the latter play an important role in shaping Manila clam microbial communities., Conclusions: Sediment management in the Venice lagoon will increase in the next years to maintain and create new canals as well as to allow the operation of the new mobile gates at the three Venice lagoon inlets. Our data reveal important transcriptional and microbial changes of Manila clams after exposure to sediments, therefore reuse of dredged sediments represents a potential risk for the conservation of this species and possibly for other organisms inhabiting the Venice lagoon., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Among veterinary antibiotics, flumequine (FLU) is still widely used in aquaculture due to its efficacy and cost-effectiveness. Although it was synthesized more than 50 years ago, a complete toxicological framework of possible side effects on non-target species is still far from being achieved. The aim of this research was to investigate the FLU molecular mechanisms in Daphnia magna , a planktonic crustacean recognized as a model species for ecotoxicological studies. Two different FLU concentrations (2.0 mg L -1 and 0.2 mg L -1 ) were assayed in general accordance with OECD Guideline 211, with some proper adaptations. Exposure to FLU (2.0 mg L -1 ) caused alteration of phenotypic traits, with a significant reduction in survival rate, body growth, and reproduction. The lower concentration (0.2 mg L -1 ) did not affect phenotypic traits but modulated gene expression, an effect which was even more evident under the higher exposure level. Indeed, in daphnids exposed to 2.0 mg L -1 FLU, several genes related with growth, development, structural components, and antioxidant response were significantly modulated. To the best of our knowledge, this is the first work showing the impact of FLU on the transcriptome of D. magna .

Aflatoxin B1 (AFB1) is a food contaminant metabolized mostly in the liver and leading to hepatic damage. Livestock species are differently susceptible to AFB1, but the underlying mechanisms of toxicity have not yet been fully investigated, especially in ruminants. Thus, the aim of the present study was to better characterize the molecular mechanism by which AFB1 exerts hepatotoxicity in cattle. The bovine fetal hepatocyte cell line (BFH12) was exposed for 48 h to three different AFB1 concentrations (0.9 µM, 1.8 µM and 3.6 µM). Whole-transcriptomic changes were measured by RNA- seq analysis, showing significant differences in the expression of genes mainly involved in inflammatory response, oxidative stress, drug metabolism, apoptosis and cancer. As a confirmatory step, post-translational investigations on genes of interest were implemented. Cell death associated with necrosis rather than apoptosis events was noted. As far as the toxicity mechanism is concerned, a molecular pathway linking inflammatory response and oxidative stress was postulated. Toll-Like Receptor 2 (TLR2) activation, consequent to AFB1 exposure, triggers an intracellular signaling cascade involving a kinase (p38β MAPK), which in turn allows the nuclear translocation of the activator protein-1 (AP-1) and NF-κB, finally leading to the release of pro-inflammatory cytokines. Furthermore, a p38β MAPK negative role in cytoprotective genes regulation was postulated. Overall, our investigations improved the actual knowledge on the molecular effects of this worldwide relevant natural toxin in cattle.

Aflatoxin B1 (AFB1) is a major food safety concern, threatening the health of humans and animals. Bentonite (BEN) is an aluminosilicate clay used as a feed additive to reduce AFB1 presence in contaminated feedstuff. So far, few studies have characterized BEN toxicity and efficacy in vitro. In this study, cytotoxicity (WST-1 test), the effects on cell permeability (trans-epithelial electrical resistance and lucifer yellow dye incorporation), and transcriptional changes (RNA-seq) caused by BEN, AFB1 and their combination (AFB1 + BEN) were investigated in Caco-2 cells. Up to 0.1 mg/mL, BEN did not affect cell viability and permeability, but it reduced AFB1 cytotoxicity; however, at higher concentrations, BEN was cytotoxic. As to RNA-seq, 0.1 mg/mL BEN did not show effects on cell transcriptome, confirming that the interaction between BEN and AFB1 occurs in the medium. Data from AFB1 and AFB1 + BEN suggested AFB1 provoked most of the transcriptional changes, whereas BEN was preventive. The most interesting AFB1-targeted pathways for which BEN was effective were cell integrity, xenobiotic metabolism and transporters, basal metabolism, inflammation and immune response, p53 biological network, apoptosis and carcinogenesis. To our knowledge, this is the first study assessing the in vitro toxicity and whole-transcriptomic effects of BEN, alone or in the presence of AFB1.

Maize root responds to nitrate by modulating its development through the coordinated action of many interacting players. Nitric oxide is produced in primary root early after the nitrate provision, thus inducing root elongation. In this study, RNA sequencing was applied to discover the main molecular signatures distinguishing the response of maize root to nitrate according to their dependency on, or independency of, nitric oxide, thus discriminating the signaling pathways regulated by nitrate through nitric oxide from those regulated by nitrate itself of by further downstream factors. A set of subsequent detailed functional annotation tools (Gene Ontology enrichment, MapMan, KEGG reconstruction pathway, transcription factors detection) were used to gain further information and the lateral root density was measured both in the presence of nitrate and in the presence of nitrate plus cPTIO, a specific NO scavenger, and compared to that observed for N-depleted roots. Our results led us to identify six clusters of transcripts according to their responsiveness to nitric oxide and to their regulation by nitrate provision. In general, shared and specific features for the six clusters were identified, allowing us to determine the overall root response to nitrate according to its dependency on nitric oxide.

There is growing concern for the wide use ofperfluorooctanoic acid (PFOA) because of its toxic effects on the environment and on human health. A new compound - the so called C6O4 (perfluoro ([5-methoxy-1,3-dioxolan-4-yl]oxy) acetic acid) - was recently introduced as one of the alternative to traditional PFOA, however this was done without any scientific evidence of the effects of C6O4 when dispersed into the environment. Recently, the Regional Agency for the Protection of the Environment of Veneto (Italy) detected high levels of C6O4 in groundwater and in the Po river, increasing the alarm for the potential effects of this chemical into the natural environment. The present study investigates for the first time the effects of C6O4 on the Manila clam Ruditapes philippinarum exposed to environmental realistic concentrations of C6O4 (0.1 µg/L and 1 µg/L) for 7 and 21 days. Furthermore, in order to better understand if C6O4 is a valid and less hazardous alternative to its substitute, microbial and transcriptomic alterations were also investigated in clams exposed to 1 µg/L ofPFOA. Results indicate that C6O4 may cause significant perturbations to the digestive gland microbiota, likely determining the impairment of host physiological homeostasis. Despite chemical analyses suggest a 5 times lower accumulation potential of C604 as compared to PFOA in clam soft tissues, transcriptional analyses reveal several alterations of gene expression profile. A large part of the altered pathways, including immune response, apoptosis regulation, nervous system development, lipid metabolism and cell membrane is the same in C6O4 and PFOA exposed clams. In addition, clams exposed to C6O4 showed dose-dependent responses as well as possible narcotic or neurotoxic effects and reduced activation of genes involved in xenobiotic metabolism. Overall, the present study suggests that the potential risks for marine organism following environmental contamination are not reduced by replacing PFOA with C6O4. In addition, the detection of both C6O4 and PFOA into tissues of clams inhabiting the Lagoon of Venice - where there are no point sources of either compounds - recommends a similar capacity to spread throughout the environment. These results prompt the urgent need to re-evaluate the use of C6O4 as it may represent not only an environmental hazard but also a potential risk for human health., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Right ventricular (RV) function impacts clinical outcomes after surgical repair of Tetralogy of Fallot (rTOF). However, assessment of RV function remains difficult. We investigated the RV myocardial systolic-to-diastolic (S/D) duration ratio derived from strain imaging time intervals to characterize RV myocardial performance, exploring its relation with peak oxygen consumption during exercise (V̇o 2 ) and cardiac magnetic resonance-derived RV dilation and function in rTOF. We retrospectively analyzed 76 children with rTOF and 42 normal controls. The RV myocardial S/D duration ratio was measured from RV global and regional 2D speckle tracking longitudinal strain. Time from QRS onset to peak systolic strain was defined as the systolic duration. The S/D duration ratio was calculated and corrected for heart rate (HR). Postsystolic shortening (PSS) duration was defined as shortening time after cessation of pulmonary systolic antegrade flow. The RV myocardial S/D duration ratio, corrected or uncorrected for HR, was significantly higher in rTOF vs. controls (1 ± 0.3 vs. 0.8 ± 0.2, P = 0.004) in relation to prolonged PSS. The HR-corrected myocardial S/D duration ratio correlated weakly with RV ejection fraction (EF, r  = -0.37, P = 0.001) and V̇o 2 ( r  = -0.32, P = 0.042). In multiregression analysis, RV EF was independently associated with the myocardial S/D duration ratio. The RV myocardial S/D duration ratio is a parameter of RV myocardial performance and efficiency, incorporating elements of systolic and diastolic performance, mechanical dyssynchrony, and PSS. The S/D duration ratio is associated with exercise capacity and RV dysfunction in rTOF. NEW & NOTEWORTHY This is the first study to assess right ventricular myocardial performance using the systolic-to-diastolic duration ratio derived from 2D strain. Seventy-six children with repaired Tetralogy of Fallot were evaluated. Echocardiographic data were correlated with cardiac magnetic resonance and peak oxygen consumption during exercise. The results show the right ventricular myocardial systolic-to-diastolic duration ratio incorporates systolic and diastolic performance, electromechanical dyssynchrony, and postsystolic shortening and is associated with exercise capacity in repaired Tetralogy of Fallot.

Glyphosate, the most widely used herbicide worldwide, targets the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme in the shikimate pathway found in plants and some microorganisms. While the potential for glyphosate to induce a broad range of biological effects in exposed organisms has been demonstrated, the global molecular mechanisms of toxicity and potential effects in bacterial symbionts remain unclear, in particular for ecologically important marine species such as bivalve molluscs. Here, the effects of glyphosate (GLY), its degradation product aminomethylphosphonic acid (AMPA), and a mixture of both (MIX) on the mussel M. galloprovincialis were assessed in a controlled experiment. For the first time, next generation sequencing (RNA-seq and 16S rRNA amplicon sequencing) was used to evaluate such effects at the molecular level in both the host and its respective microbiota. The results suggest that the variable capacity of bacterial species to proliferate in the presence of these compounds and the impairment of host physiological homeostasis due to AMPA and GLY toxicity may cause significant perturbations to the digestive gland microbiota, as well as elicit the spread of potential opportunistic pathogens such as Vibrio spp.. The consequent host-immune system activation identified at the molecular and cellular level could be aimed at controlling changes occurring in the composition of symbiotic microbial communities. Overall, our data raise further concerns about the potential adverse effects of glyphosate and AMPA in marine species, suggesting that both the effects of direct toxicity and the ensuing changes occurring in the host-microbial community must be taken into consideration to determine the overall ecotoxicological hazard of these compounds., Competing Interests: Declaration of 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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mass mortalities due to disease outbreaks have recently affected a number of major taxa in marine ecosystems. Climate- and pollution-induced stress may compromise host immune defenses, increasing the risk of opportunistic diseases. Despite growing evidence that mass mortality events affecting marine species worldwide are strongly influenced by the interplay of numerous environmental factors, the reductionist approaches most frequently used to investigate these factors hindered the interpretation of these multifactorial pathologies. In this study, we propose a broader approach based on the combination of RNA-sequencing and 16S microbiota analyses to decipher the factors underlying mass mortality in the striped venus clam, Chamelea gallina, along the Adriatic coast. On one hand, gene expression profiling and functional analyses of microbial communities showed the over-expression of several genes and molecular pathways involved in xenobiotic metabolism, suggesting potential chemical contamination in mortality sites. On the other hand, the down-regulation of several genes involved in immune and stress response, and the over-representation of opportunistic pathogens such as Vibrio and Photobacterium spp. indicates that these microbial species may take advantage of compromised host immune pathways and defense mechanisms that are potentially affected by chemical exposure, resulting in periodic mortality events. We propose the application of our approach to interpret and anticipate the risks inherent in the combined effects of pollutants and microbes on marine animals in today's rapidly changing environment., (© 2019 John Wiley & Sons Ltd.)

Objective: To reduce the ovarian dose with volumetric modulated arc therapy (VMAT), an original VMAT was designed with two types of arcs to restrict angles and fields (R-VMAT)., Methods: The subjects were 11 patients who underwent ovarian transposition with clips left by a surgeon. Three methods, intensity-modulated radiotherapy (IMRT), standard VMAT (S-VMAT) and R-VMAT, were optimized for assessment of the ovarian dose with the target coverage kept high., Results: The homogeneity and conformity indexes for the planning target volume (PTV) were similar for the three methods. However, the average ± SD of the ovarian mean dose (OMD) was 5.0 ± 1.5, 4.9 ± 1.9 and 3.5 ± 1.4 Gy, and the percentage of ovarian volume exceeding 5 Gy (V5) was 41.5 ± 34.1%, 34.1 ± 38.05% and 8.4 ± 20.5% for IMRT, S-VMAT and R-VMAT, respectively. The OMD and V5 were significantly smaller for R-VMAT than for the other plans (p < 0.01). Correlation values between the OMD and the lateral distance from the ovaries to the PTV surface were 0.86, 0.81 and 0.82 for IMRT, S-VMAT and R-VMAT, respectively., Conclusion: These findings suggest that R-VMAT delivered the lowest dose to the ovaries. To reduce the OMD to less than 3 Gy, ovaries should be transposed laterally 6.1 cm away from the PTV surface when R-VMAT is used. Advances in knowledge: When organs with high susceptibility to radiation, such as ovaries, are near the PTV, R-VMAT is superior to IMRT and S-VMAT.

Purpose: The purpose of the present study was to retrospectively evaluate the outcomes of patients who underwent combined medial unicompartmental knee arthroplasty (UKA) and anterior cruciate ligament (ACL) reconstruction. The hypothesis was that this procedure would lead to a high success rate in patients affected by isolated medial unicompartmental osteoarthritis and concomitant ACL deficiency., Methods: Fourteen patients with primary ACL lesion and concomitant medial compartment symptomatic osteoarthritis treated from 2006 to 2010 were followed up for an average time of 26.7 months (SD 4.2). Assessment included KOOS score, Oxford Knee score, American Knee Society scores, WOMAC index of osteoarthritis, Tegner activity level and objective examination including instrumented laxity test with KT-1000 arthrometer. Radiological assessment was done with standard simple radiographs in order to get information about any presence of loosening of the components., Results: KOOS score, OKS, WOMAC index and the AKSS improved significantly after surgery (p < 0.001). Regarding AKSS, improvement was noted both in the objective score and in the functional one (p < 0.001). There was no clinical evidence of instability in any of the knees as evaluated with clinical laxity testing. No pathologic radiolucent lines were observed around the components. In one patient signs of osteoarthritis in the lateral compartment were observed 28 months after surgery., Conclusions: UKA combined with ACL reconstruction is a valid therapeutic option for the treatment of combined medial unicompartmental knee osteoarthritis and ACL deficiency in young and active patients and confirms subjective and objective clinical improvement 2 years after surgery. The use of a fixed-bearing prosthesis represents a reliable feature as it allows to overcome problems of improper ligament tensioning during the implantation of the components., Level of Evidence: IV.

Purpose: Second- and third-generation cementless Zweymüller stems have achieved good long-term outcomes in total hip arthroplasty (THA). The PPF cementless proximal press-fit femoral stem (Biomet Inc.) is based on the Zweymüller stem, with some prominent design differences. This study was designed to assess its performance in terms of survivorship, clinical function, and radiographic outcomes., Methods: We retrospectively analysed 250 patients (303 hips) who underwent THA between 1998 and 2011. Patients attended a follow-up visit a mean 10.9 ± 2.6 years after the procedure. All patients underwent radiographic examination and were clinically assessed for Forgotten Joint Score (FJS) and pain visual analogue scale (VAS). Survivorship was calculated using Kaplan-Meier analysis., Results: Clinical data were available for 142 patients (192 hips) and radiographic data for 79 patients (109 hips). Stem survival was 99.1% (95% confidence interval [CI] 96.6%-99.8%) at 10 years and 95.6% (95% CI 96.6%-99.8%) at 15 years. The median postoperative FJS was 91 (interquartile range [IQR], 75-100). The median VAS score was 0 (IQR, 0-2). Radiolucent lines were observed in 47 hips (43.1%). 25 hips (22.9%) had Grade I ossification, 17 (15.6%) Grade II, and 2 (1.8%) Grade III. 12 hips (11.0%) had osteolysis in at least 1 femoral zone., Conclusions: The cementless proximal press-fit femoral stem was associated with good mid- to long-term survival and functional outcomes. The current design can be considered equivalent to the second- and third-generation Zweymüller stems.

Purpose: The study was designed to compare the clinical results of traditional single-bundle (SB) anterior cruciate ligament (ACL) reconstruction with those of double-bundle (DB) ACL reconstruction., Methods: This study comprised 80 patients aged 18 to 45 years with an isolated ACL lesion: 40 patients underwent SB reconstruction, and 40 patients underwent DB reconstruction. Patients were assessed preoperatively with functional assessment including the International Knee Documentation Committee 2000 knee subjective form and visual analog scale, as well as physical examination (including the pivot-shift test and instrumented knee laxity measurement). Vertical jump assessment with the Optojump system (Microgate, Bolzano, Italy) has been introduced as a method to compare functional ability between the 2 surgical techniques. The same protocol was repeated 6 months, 12 months, and 2 years after surgery., Results: No statistically significant differences were noted between the groups concerning subjective evaluation, thigh girth difference, mean visual analog scale score, range of motion, and Lachman and anterior drawer tests (P = not significant). A statistically higher number of patients in the SB group showed a positive pivot-shift test and a higher side-to-side difference when measured with the KT-1000 arthrometer (MEDmetric, San Diego, CA) than in the DB group (P < .001). Better mean jumping performance results were reported in the DB group compared with the SB group (P < .001). The average performance results for the injured limb were not significantly reduced compared with those of the uninjured limb in the DB group 12 months after surgery. At 2 years, a restoration of jumping ability in the ACL-reconstructed limb was achieved in both groups regardless of the technique used., Conclusions: DB ACL reconstruction has been proven to be superior to the SB technique with regard to knee stability and vertical jump performance., Level of Evidence: Level II, prospective comparative study., (Copyright © 2013 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Purpose: This study was conducted to evaluate the role of computed tomography cystography (CTC) and virtual cystoscopy (VC) with 64-slice CT in diagnosing bladder lesions using flexible cystoscopy as the reference standard., Materials and Methods: Twenty-eight patients with suspected bladder cancer and ten patients who had undergone transurethral resection of the bladder were studied by CTC and VC in both the supine and prone positions after distending the bladder with air. The patient population was divided into three groups based on lesion size at flexible cystoscopy. Results of the CT study were compared with those of flexible cystoscopy., Results: Flexible cystoscopy depicted 88 bladder lesions in the 38 patients examined. Sensitivity and specificity values of CTC and VC alone were constantly lower than those of the combined-approach (group 1: 93.10% and 92.31%; group 2: 100% and 100%; group 3: 100% and 100%, respectively). Receiver operating characteristic (ROC) curve analysis showed that the combined approach decreases the lower dimensional threshold for lesion detection (1.4 mm)., Conclusions: VC and CTC with 64-slice CT are promising diagnostic techniques for bladder cancers measuring 1-5 mm. Further studies on larger patient populations are required to validate our results.

Our objective was to optimize a scanning protocol for CT colonography using a multidetector scanner with an adaptive-array matrix in a phantom study. A colonic phantom with 27 simulated lesions was examined using a multidetector CT scanner (Somatom Plus 4 Volume Zoom, Siemens, Erlangen, Germany). Three scanning protocols were tested: (a) collimation 1.0 mm, slice thickness 1.0 mm; (b) collimation 2.5 mm, slice thickness 3.0 mm; and (c) collimation 2.5 mm, slice thickness 5.0 mm. Image analysis was performed by two radiologists blinded to the construction of the phantom and to imaging parameters. Sensitivity for lesion detection and image quality was assessed. Computed tomographic colonography detected 27 of 27 lesions with protocol 1 (sensitivity 100%); 26 of 27 lesions with protocol 2 (sensitivity 96.3%); and 23 of 27 lesions with protocol 3 (sensitivity 85.2%). Image quality was graded as optimal for protocol 1, good for protocol 2, and poor for protocol 3. When multidetector CT equipment with an adaptive-array matrix is used, protocols with 1.0- or 3.0-mm slice thickness have a sensitivity for the detection of lesions higher than that obtained with a 5.0-mm slice thickness. Image quality decreases progressively when the slice thickness is increased; therefore, in a clinical setting, we recommend the use of a high-resolution protocol (1.0-mm effective slice thickness) with the highest pitch value in order to reduce scanning time. Dose exposure for such a protocol if combined with 80 mAs is within the range limits recommended by the ICRP for abdominal CT scanning.

The aim of the present study was to assess the role of virtual cystoscopy in the identification of bladder tumors. Fifteen patients (11 men and 4 women, median age: 61 years, range: 46-74 years) with a positive finding of bladder tumor at fiber-optic cystoscopy were studied by multislice-CT. Scans were downloaded to a workstation with the aid of a software for the processing of 3-D reconstructions, with a volume-rendering technique which allowed the "navigation" within the bladder in search of wall lesions. In this group of 15 patients, cystoscopy was able to detect 19 neoplastic lesions, 13 with a diameter >10 mm and 6 with a diameter <10 mm. Virtual cystoscopy, instead, identified 17 lesions (89%) only. In particular, all those lesions with a diameter >1 cm (13/13=100%) were correctly identified, whereas only 4 of the 6 lesions with a diameter <1 cm were depicted. The 2 false negative cases were 2 lesions with a flat morphology, measuring 5 and 6 mm. Most recent technological advances allowed the employement of virtual endoscopies, characterized by the absence of invasivity as compared with fiber-optic studies and based on data obtained by spiral- and multislice-CTs. According to our experience, virtual CT-cystoscopy revealed to be a complementary tool in the evaluation of cross-sectional images and proved to be an easy procedure without complications, well-accepted by the patients, and with a reliable detection of those bladder lesions measuring more than 5 mm in case of polypoid formations and at least 10 mm in case of flat lesions. This technique, however, does not allow the collection of a bioptic sample and--with the present resolution power of available equipments--it could be unable to correctly detect small-sized flat lesions. We, nonetheless, believe that this procedure, in the future, thanks to rapid technological improvements in virtual imaging techniques, could become a useful diagnostic tool in the management of those patients with bladder tumors. Further studies on larger study groups are therefore desirable for a more reliable validation of the technique.

Purpose: To optimize scanning parameters for virtual colonoscopy utilizing a multislice Helical CT scanner in an in vitro study (using a homemade colonic phantom) and in a preliminary clinical study., Material and Methods: A colonic phantom was built using a plastic tube and 12 plastiline polyps were placed inside. The colonic phantom was studied with a multislice Helical CT scanner. Axial images were obtained with the phantom parallel to the long axis of the moving table (in order to simulate the evaluation of ascending and descending colon): oblique images were acquired with the phantom at 45 degrees relative to the long axis of the moving table (in order to simulate the evaluation of sigmoid colon and colonic flexures). Four different scanning protocols were tested: 1) slice collimation, 5 mm; slice width, 7 mm; table speed, 25 mm; reconstruction index, 5 mm; 2) slice collimation, 2.5 mm; slice width, 3 mm; table speed, 15 mm; reconstruction index, 3 mm; 3) slice collimation, 1 mm; slice width, 1.25 mm; table speed, 5 mm; reconstruction index, 1 mm; 4) slice collimation, 1 mm; slice width, 1.25 mm; table speed, 4 mm; reconstruction index, 1 mm. Quantitative analysis consisted in evaluation of the number of identified polyps and polyp size along the longitudinal axis. Qualitative analysis consisted in the evaluation of image artifacts and quality of 3D reconstructed images (step artifacts and polyp geometry distortion). This preliminary clinical study was performed in 12 patients (7 men and 5 women) who underwent multislice Helical CT colonography. We selected patients with clinical indications for conventional colonoscopy or after unsuccessful conventional colonoscopy., Results: Multislice Helical CT colonography was 100% sensitive in the detection of all polyps and in all scanning protocols. With oblique scans, only a 3-mm polyp was missed during protocol 1 (sensitivity: 92%). Polyp geometry distortion was observed on longitudinal reconstructions, whereas no distortion was seen on axial images. Image quality was graded as optimal for protocols 2, 3, and 4; protocol 1 was graded as good on transverse scans and as poor on oblique scans. In our preliminary clinical study, two colonic carcinomas and three polyps were identified., Conclusions: At present, the introduction of multislice technology in virtual colonoscopy permits to improve spatial resolution and image definition. The actual clinical advantage, in terms of increased diagnostic accuracy, needs further investigation in larger clinical studies.

The purpose of this study was to investigate the effect of carvedilol on serum lipids in patients with mild-to-moderate essential hypertension. Twenty-one patients with blood pressure greater than or equal to 160/95 mm Hg after a 4-week placebo run-in period were initially given 10 mg of carvedilol once daily. The dose was increased to 20 mg after 4 weeks if the target blood pressure was not achieved. The duration of treatment was 12 weeks. After 12 weeks of administration, blood pressure and the pulse rate (PR) declined significantly (blood pressure from 173/105 to 142/91 mm Hg, p less than 0.001; PR from 74 to 67 beats/min, p less than 0.001); however, serum lipids [total cholesterol, triglycerides, low-density lipoprotein, high-density lipoprotein (HDL), HDL2, and HDL3], lipoprotein fraction (alpha, pre-beta, and beta), apoprotein fraction (A-I, A-II, CII, CIII, and E), and atherogenic index [(total cholesterol - HDL cholesterol) divided by HDL cholesterol] were not altered significantly. There were no side effects reported during the trial. From these results, it can be concluded that carvedilol has no adverse effect on the coronary risk profile as reflected by lipid measurements, and is an efficacious, safe, well-tolerated antihypertensive drug in patients with mild-to-moderate hypertension.