Perinatal outcomes in lesbian couples employing shared motherhood IVF compared with those performing artificial insemination with donor sperm.

STUDY QUESTION: In lesbian couples, is shared motherhood IVF (SMI) associated with an increase in perinatal complications compared with artificial insemination with donor sperm (AID)? SUMMARY ANSWER: Singleton pregnancies in SMI and AID had very similar outcomes, except for a non-significant increase in the rate of preeclampsia/hypertension (PE/HT) in SMI (recipient's age-adjusted odds ratio (OR) = 1.9, 95% CI = 0.7-5.2; P = 0.19), but twin SMI pregnancies had a much higher frequency of PE/HT than AID twins (recipient's age-adjusted OR = 21.7, 95% CI = 2.8-289.4; P = 0.01). WHAT IS KNOWN ALREADY: Oocyte donation (OD) pregnancies are associated with an increase in perinatal complications, in particular, preterm delivery and low birth weight, and PE/HT. However, it is unclear to what extent these complications are due to OD process or to the conditions why OD was performed, such as advanced age and underlying health conditions. Unfortunately, the literature concerning perinatal outcomes in SMI is scarce. STUDY DESIGN, SIZE, DURATION: Retrospective study involving 660 SMI cycles (299 pregnancies) and 4349 AID cycles (949 pregnancies) assisted over a 10-year period. PARTICIPANTS/MATERIALS, SETTING, METHODS: All cycles fulfilling the inclusion criteria performed in lesbian couples seeking fertility treatment in 17 Spanish clinics of the same group. Pregnancy rates of SMI and AID cycles were compared. Perinatal outcomes were compared: gestational length, newborn weight, preterm and low birth rates, PE/HT rates, cesarean section rates, perinatal mortality, and newborn malformations. MAIN RESULTS AND THE ROLE OF CHANCE: Pregnancy rates were higher in SMI than in AID (45.3% versus 21.8%, P < 0.001). There was a non-significant trend to higher multiple rate in AID (4.7% versus 8.5%, P = 0.08). In single pregnancies, there were no differences between SMI and AID in gestational age (278 days (268-285) versus 279 (272-284), P = 0.24), preterm rate (8.3% versus 7.3%, P = 0.80), preterm <28 weeks (0.6% versus 0.4%, P = 1.00), newborn weight (3195 g (2915-3620) versus 3270 g (2980-3600), P = 0.296), low birth rate (6.4% versus 6.4%, P = 1.00), extremely low birth weight (0.6% versus 0.5%, P = 1.00), and the distribution of newborns by weight groups. Cesarean section rate, newborn malformation rate, and perinatal mortality were also similar in SMI and AID. Additionally, there was non-significant trend in hypertensive disorders to an increase in PE/HT among SMI (recipient's age-adjusted OR = 1.9, 95% CI = 0.7-5.2). Overall, perinatal data are consistent with what is reported in the general population. In twin pregnancies, the aforementioned perinatal parameters were also very similar in SMI and AID. However, SMI twin pregnancies had a very high risk of PE/HT when compared with AID (recipient's age-adjusted OR = 21.7, 95% CI = 2.8-289.4, P = 0.01). LIMITATIONS, REASONS FOR CAUTION: Our data regarding the pregnancy course were obtained from information registered in the delivery report as well as from what was reported by the patients themselves, so a certain degree of inaccuracy cannot be ruled out. Additionally, in some parameters, there was up to 10% of data missing. However, since the methodology of reporting was the same in SMI and AID groups, one should not expect a differential reporting bias. It cannot be ruled out that the risk of PE/HT in simple gestations would be significant in a larger study. Additionally, in the SMI group allocation to the transfer of 2 embryos was not randomized so some bias is possible. WIDER IMPLICATIONS OF THE FINDINGS: SMI, if single embryo transfer is performed, seems to be is a safe procedure. Double embryo transfer should not be performed in SMI. Our data suggest that the majority of complications in OD could be related more with recipient status than with OD itself, since with SMI (performed in women without fertility problems) the perinatal complications were much lower than usually described in OD. STUDY FUNDING/COMPETING INTEREST(S): No external funding was received. The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

'Value-based methodology for person-centred, integrated care supported by Information and Communication Technologies' (ValueCare) for older people in Europe: study protocol for a pre-post controlled trial.

BACKGROUND: Older people receive care from multiple providers which often results in a lack of coordination. The Information and Communication Technology (ICT) enabled value-based methodology for integrated care (ValueCare) project aims to develop and implement efficient outcome-based, integrated health and social care for older people with multimorbidity, and/or frailty, and/or mild to moderate cognitive impairment in seven sites (Athens, Greece; Coimbra, Portugal; Cork/Kerry, Ireland; Rijeka, Croatia; Rotterdam, the Netherlands; Treviso, Italy; and Valencia, Spain). We will evaluate the implementation and the outcomes of the ValueCare approach. This paper presents the study protocol of the ValueCare project; a protocol for a pre-post controlled study in seven large-scale sites in Europe over the period between 2021 and 2023. METHODS: A pre-post controlled study design including three time points (baseline, post-intervention after 12 months, and follow-up after 18 months) and two groups (intervention and control group) will be utilised. In each site, (net) 240 older people (120 in the intervention group and 120 in the control group), 50-70 informal caregivers (e.g. relatives, friends), and 30-40 health and social care practitioners will be invited to participate and provide informed consent. Self-reported outcomes will be measured in multiple domains; for older people: health, wellbeing, quality of life, lifestyle behaviour, and health and social care use; for informal caregivers and health and social care practitioners: wellbeing, perceived burden and (job) satisfaction. In addition, implementation outcomes will be measured in terms of acceptability, appropriateness, feasibility, fidelity, and costs. To evaluate differences in outcomes between the intervention and control group (multilevel) logistic and linear regression analyses will be used. Qualitative analysis will be performed on the focus group data. DISCUSSION: This study will provide new insights into the feasibility and effectiveness of a value-based methodology for integrated care supported by ICT for older people, their informal caregivers, and health and social care practitioners in seven different European settings. TRIAL REGISTRATION: ISRCTN registry number is 25089186 . Date of trial registration is 16/11/2021.

Identifying and optimizing human endometrial gene expression signatures for endometrial dating.

STUDY QUESTION: What are the key considerations for developing an enhanced transcriptomic method for secretory endometrial tissue dating? SUMMARY ANSWER: Multiple gene expression signature combinations can serve as biomarkers for endometrial dating, but their predictive performance is variable and depends on the number and identity of the genes included in the prediction model, the dataset characteristics and the technology employed for measuring gene expression. WHAT IS KNOWN ALREADY: Among the new generation of transcriptomic endometrial dating (TED) tools developed in the last decade, there exists variation in the technology used for measuring gene expression, the gene makeup and the prediction model design. A detailed study, comparing prediction performance across signatures for understanding signature behaviour and discrepancies in gene content between them, is lacking. STUDY DESIGN, SIZE, DURATION: A multicentre prospective study was performed between July 2018 and October 2020 at five different centres from the same group of clinics (Spain). This study recruited 281 patients and finally included in the gene expression analysis 225 Caucasian patients who underwent IVF treatment. After preprocessing and batch effect filtering, gene expression measurements from 217 patients were combined with artificial intelligence algorithms (support vector machine, random forest and k-nearest neighbours) allowing evaluation of different prediction models. In addition, secretory-phase endometrial transcriptomes from gene expression omnibus (GEO) datasets were analysed for 137 women, to study the endometrial dating capacity of genes independently and grouped by signatures. This provided data on the consistency of prediction across different gene expression technologies and datasets. PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometrial biopsies were analysed using a targeted TruSeq (Illumina) custom RNA expression panel called the endometrial dating panel (ED panel). This panel included 301 genes previously considered relevant for endometrial dating as well as new genes selected for their anticipated value in detecting the secretory phase. Final samples (n = 217) were divided into a training set for signature discovery and an independent testing set for evaluation of predictive performance of the new signature. In addition, secretory-phase endometrial transcriptomes from GEO were analysed for 137 women to study endometrial dating capacity of genes independently and grouped by signatures. Predictive performance among these signatures was compared according to signature gene set size. MAIN RESULTS AND THE ROLE OF CHANCE: Testing of the ED panel allowed development of a model based on a new signature of 73 genes, which we termed 'TED' and delivers an enhanced tool for the consistent dating of the secretory phase progression, especially during the mid-secretory endometrium (3-8 days after progesterone (P) administration (P + 3-P + 8) in a hormone replacement therapy cycle). This new model showed the best predictive capacity in an independent test set for staging the endometrial tissue in the secretory phase, especially in the expected window of implantation (average of 114.5 ± 7.2 h of progesterone administered; range in our patient population of 82-172 h). Published sets of genes, in current use for endometrial dating and the new TED genes, were evaluated in parallel in whole-transcriptome datasets and in the ED panel dataset. TED signature performance was consistently excellent for all datasets assessed, frequently outperforming previously published sets of genes with a smaller number of genes for dating the endometrium in the secretory phase. Thus, this optimized set exhibited prediction consistency across datasets. LARGE SCALE DATA: The data used in this study is partially available at GEO database. GEO identifiers GSE4888, GSE29981, GSE58144, GSE98386. LIMITATIONS, REASONS FOR CAUTION: Although dating the endometrial biopsy is crucial for investigating endometrial progression and the receptivity process, further studies are needed to confirm whether or not endometrial dating methods in general are clinically useful and to guide the specific use of TED in the clinical setting. WIDER IMPLICATIONS OF THE FINDINGS: Multiple gene signature combinations provide adequate endometrial dating, but their predictive performance depends on the identity of the genes included, the gene expression platform, the algorithms used and dataset characteristics. TED is a next-generation endometrial assessment tool based on gene expression for accurate endometrial progression dating especially during the mid-secretory. STUDY FUNDING/COMPETING INTEREST(S): Research funded by IVI Foundation (1810-FIVI-066-PD). P.D.-G. visiting scientist fellowship at Oxford University (BEFPI/2010/032) and Josefa Maria Sanchez-Reyes' predoctoral fellowship (ACIF/2018/072) were supported by a program from the Generalitat Valenciana funded by the Spanish government. A.D.-P. is supported by the FPU/15/01398 predoctoral fellowship from the Ministry of Science, Innovation and Universities (Spanish Government). D.W. received support from the NIHR Oxford Biomedical Research Centre. The authors do not have any competing interests to declare.

Estrogen Replacement Therapy Induces Antioxidant and Longevity-Related Genes in Women after Medically Induced Menopause.

Females live longer than males in many species, including humans, and estrogens are in part responsible for this protection against aging. We reported previously that estrogens can protect rats against oxidative stress, by inducing antioxidant and longevity-related genes. Thus, this study was aimed at confirming the ability of estrogens to upregulate antioxidant and longevity-related genes in humans. For this purpose, we selected 16 women of reproductive age (18-42 years old) undergoing a fertility treatment that includes a medically induced menopause, at the Valencian Infertility Institute. We took blood samples at each time point of the treatment (basal, induced menopause, estrogen, and estrogen plus progesterone replacement therapy). mRNA expression of antioxidant and longevity-related genes in peripheral blood mononuclear cells (PBMC) was determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Determination of reduced glutathione (GSH) in total blood was carried out using high-performance liquid chromatography (HPLC). As expected, we found that medically induced menopause significantly decreased sexual hormone (estrogens and progesterone) levels. It also lowered glutathione peroxidase (GPx), 16S rRNA, P21, and TERF2 mRNA expression and blood GSH levels. Estrogen replacement therapy significantly restored estrogen levels and induced mRNA expression of manganese superoxide dismutase (MnSOD), GPx, 16S rRNA, P53, P21, and TERF2 and restored blood GSH levels. Progesterone replacement therapy induced a significant increase in MnSOD, P53, sestrin 2 (SENS2), and TERF2 mRNA expression when compared to basal conditions. These findings provide evidence for estrogen beneficial effects in upregulating antioxidant and longevity-related genes in women.

The prevalence of sleep impairments and predictors of sleep quality among patients with asthma.

BACKGROUND: Sleep is a significant dimension of daily life. However, only a few studies have examined the sleep quality of asthmatics in a real-world clinical settings. OBJECTIVE: This study is aimed to estimate the prevalence of sleep impairments among asthmatic patients and examine the relationship between sleep quality, asthma control, rhinitis symptoms, and sociodemographic characteristics. METHODS: The present study adopted the observational cross-sectional research design that has been designed by the Italian Respiratory Society and used valid assessments to measure the study variables. RESULTS: Data from 1150 asthmatic patients (mean age 51.01 years ± 16.03) were subjected to analysis. 58.3% of the patients had impaired sleep quality (Pittsburgh Sleep Quality Index [PSQI] total scores > 5), and their mean PSQI score was 5.68 (SD = 3.4). A significant correlation emerged between sleep quality and asthma control (p = 0.0001) and a significant albeit weak correlation emerged between PSQI total scores and Total 5 Symptoms Score (r = 0.24, p = 0.0001). Sleep quality was significantly associated health-related quality of life [HRQoL]. (r = 0.50, p < 0.001). After exclusion of patients at risk for Obstructive Sleep Apnea Syndrome (OSAS) and Gastro Esophageal Reflux Disease (GERD), the most important determinants of PSQI score were HRQoL, In the entire sample asthma control is the strongest predictor of both sleep quality and HRQoL. CONCLUSIONS: The results of this real-world study highlight the prevalence, impact and predictors of sleep disturbances in asthmatic patients and suggest the need for physicians to detect poor sleep quality.

Prevalence of diabetes, use of antidiabetic drugs and degree of glycaemic control in the province of Cadiz (2014-2016): A population-based study. / Prevalencia de diabetes, utilización de antidiabéticos y grado de control glucémico en la provincia de Cádiz (2014-2016): un estudio de base poblacional.

BACKGROUND AND OBJECTIVE: The aim of the study was to assess the prevalence of diabetes mellitus treated pharmacologically, analyse the prescription patterns of antidiabetic drugs and assess the degree of control over the disease in the province of Cadiz. PATIENTS AND METHODS: An observational retrospective study was conducted with the databases of the public health system of the Andalusian Health Service between 2014 and 2016, inclusive. Adults with treated diabetes (ATD) were considered those older than 14 years who had consumed at least 1 package of medication from the A10 group during the corresponding year covered by the study. RESULTS: The prevalence of ATD varied between 8.65% and 8.83% from 2014 to 2016, respectively. Seventy-one percent of the ATD were treated with only noninsulin drugs, 11% were treated with insulin, and 18% were treated with a combination of both. For approximately one-third of the ATD, an HbA1c reading was not performed during each year. Sixty-nine percent of the assessed ATD in 2016 had an appropriate degree of control according to RedGDPS criteria (based on HbA1c and age). CONCLUSION: The prevalence of pharmacologically treated diabetes in the province of Cadiz is high and appears to be increasing. The patients presented limited glycaemic control, to which inadequate follow-up in almost a third of the patients could be the major contributor.

Nasal cytology: Methodology with application to clinical practice and research.

Nasal cytology is an easy, cheap, non-invasive and point-of-care method to assess nasal inflammation and disease-specific cellular features. By means of nasal cytology, it is possible to distinguish between different inflammatory patterns that are typically associated with specific diseases (ie, allergic and non-allergic rhinitis). Its use is particularly relevant when other clinical information, such as signs, symptoms, time-course and allergic sensitizations, is not enough to recognize which of the different rhinitis phenotypes is involved; for example, it is only by means of nasal cytology that it is possible to distinguish, among the non-allergic rhinitis, those characterized by eosinophilic (NARES), mast cellular (NARMA), mixed eosinophilic-mast cellular (NARESMA) or neutrophilic (NARNE) inflammation. Despite its clinical usefulness, cheapness, non-invasiveness and easiness, nasal cytology is still underused and this is at least partially due to the fact that, as far as now, there is not a consensus or an official recommendation on its methodological issues. We here review the scientific literature about nasal cytology, giving recommendations on how to perform and interpret nasal cytology.

Assessment of chronic effects of tebuconazole on survival, reproduction and growth of Daphnia magna after different exposure times.

The effect of the fungicide tebuconazole (0.41, 0.52, 0.71 and 1.14mg/L) on survival, reproduction and growth of Daphnia magna organisms was monitored using 14 and 21 days exposure tests. A third experiment was performed by exposing D. magna to the fungicide for 14 days followed by 7 days of recovery (14+7). In order to test fungicide effects on D. magna, parameters as survival, mean whole body length, mean total number of neonates per female, mean number of broods per female, mean brood size per female, time to first brood/reproduction and intrinsic rate of natural increase (r) were used. Reproduction was seriously affected by tebuconazole. All tebuconazole concentrations tested affected the number of broods per female and day to first brood. At 14-days test, number of neonates per female and body size decreased by concentrations of tebuconazole higher than 0.52mg/L, whereas at 21-days test both parameters were affected at all the concentrations tested. Survival of the daphnids after 14 days fungicide exposure did not exhibited differences among experimental and control groups. In this experiment r value was reduced (in a 22%) when animals were exposed to concentrations of 0.71mg/L and 1.14mg/L. Survival of daphnids exposed during 21 days to 1.14mg/L declined, and the intrinsic rate of natural increase (r) decreased in a 30 % for tebuconazole concentrations higher than 0.41mg/L. Longevity of daphnids pre-exposed to tebuconazole for 14 days and 7 days in clean water did not show differences from control values and all of them survived the 21 days of the test. However, after 7 days in fungicide free medium animals were unable to restore control values for reproductive parameters and length. The maximum acceptable toxicant concentration (MATC) was calculated using the r values as parameter of evaluation. MATC estimations were 0.61mg/L and 0.46mg/L for 14 and 21 days, respectively. Results showed that the number of neonates per female was the highest sensitive parameter to the effects of tebuconazole on D. magna. On the other hand, a recovery period of 7 days in a free toxicant medium would not be longer enough to reestablish normal reproduction parameters in pre-exposed tebuconazole daphnids.

Energy reserves mobilization in the yellow eel as herbicide exposure effect.

Thiobencarb and propanil are two of the most extensive used herbicides worldwide in rice cultivation. Especially scanty is the available information regarding the effect of herbicides on fish energy resources. In the present study, the effect of sublethal exposure to these herbicides on the energy reserves of juvenile eel Anguilla anguilla was compared. Eels were exposed to 72 h to the herbicide thiobencarb (0.22 mg L(-1)) or Propanil (0.63 mg L(-1)), and allowed to recover in clean water (144 h). Caloric content was determined in liver and skeletal muscle. Fish exposed to thiobencarb rapidly mobilized energy. Reserves from liver were depleted (21%) compared to control values (2.50 kcal g(-1)) at 2 h, whereas in muscle diminished between 12 and 72 h (35%) (control value 0.89 kcal g(-1)). Energy reserves from liver normalized after 144 h in water while in the skeletal muscle were still depleted (24%). Major harmful effects were induced by propanil. Caloric content in liver diminished from the first hours (depletion of 70% at 48h exposure) and in skeletal muscle a 60% (72 h). At the end of the recovery period, energy reserves in pre-exposed eels represented less than 50% compared to control animals. The study indicated that thiobencarb and propanil would constitute a great risk to animals inhabiting freshwater bodies nearby fields of application. Judging from the results, herbicides resulted toxic enough to mobilize fish energy stores. On the other hand, a period of six days in herbicide-free water was not enough time to allow fish to restore energy budgets.

Effects of the acupoints PC 6 Neiguan and LR 3 Taichong on cerebral blood flow in normal subjects and in migraine patients.

Acupuncture has been proven to be effective in the treatment of various cardiovascular disorders; it acts both on the peripheral flow and on the cerebral flow. Our study aimed to evaluate the effects of the insertion of PC 6 Neiguan and LR 3 Taichong acupoints on the cerebral blood flow (CBF) in the middle cerebral artery (MCA). These effects were measured in a group of patients suffering from migraine without aura (Group M) and in a healthy control group (Group C). In the study, we included 16 patients suffering from migraine without aura, classified according to the criteria of the International Headache Society, and 14 healthy subjects as a control group. The subjects took part in the study on two different days, and on each day, the effect of a single acupoint was evaluated. Transcranial Doppler was used to measure the blood flow velocity (BFV) in the MCA. Our study showed that the stimulation of PC 6 Neiguan in both groups results in a significant and longlasting reduction in the average BFV in the MCA. After pricking LR 3 Taichong, instead, the average BFV undergoes a very sudden and marked increase; subsequently, it decreases and tends to stabilize at a slightly higher level compared with the baseline, recorded before needle insertion. Our data seem to suggest that these two acupoints have very different effects on CBF. The insertion of PC 6 Neiguan probably triggers a vasodilation in MCA, while the pricking of LR 3 Taichong determines a rapid and marked vasoconstriction.

Carbohydrate availability regulates virulence gene expression in Streptococcus suis.

Streptococcus suis is a major bacterial pathogen of young pigs causing worldwide economic problems for the pig industry. S. suis is also an emerging pathogen of humans. Colonization of porcine oropharynx by S. suis is considered to be a high risk factor for invasive disease. In the oropharyngeal cavity, where glucose is rapidly absorbed but dietary α-glucans persist, there is a profound effect of carbohydrate availability on the expression of virulence genes. Nineteen predicted or confirmed S. suis virulence genes that promote adhesion to and invasion of epithelial cells were expressed at higher levels when S. suis was supplied with the α-glucan starch/pullulan compared to glucose as the single carbon source. Additionally the production of suilysin, a toxin that damages epithelial cells, was increased more than ten-fold when glucose levels were low and S. suis was growing on pullulan. Based on biochemical, bioinformatics and in vitro and in vivo gene expression studies, we developed a biological model that postulates the effect of carbon catabolite repression on expression of virulence genes in the mucosa, organs and blood. This research increases our understanding of S. suis virulence mechanisms and has important implications for the design of future control strategies including the development of anti-infective strategies by modulating animal feed composition.

Caloric content of Daphnia magna as reflect of propanil stress during a short-term exposure and its relationship to long-term responses.

The present study investigates energy stores changes in the aquatic invertebrate Daphnia magna following a 5-d exposure to propanil. Juveniles of D. magna were exposed to sublethal propanil concentrations (0.07, 0.10, 0.21 and 0.55 mgl(-1)) which were used previously to test their effect on reproduction, growth and survival (21 days test) of D. magna. Glycogen, total lipids, proteins, and dry weight were determined in control and exposed daphnids at 24, 48, 72, 96 and 120 h. Data were used to calculate caloric content as biomarker of propanil exposure. Results showed a depletion of energy reserves in D. magna exposed to the herbicide. At 120 h of exposure to the highest propanil concentration (0.5 mgl(-1)), the reduction in daphnid reserves were glycogen (28%), proteins (28%) and lipids (31%), and caloric content was reduced in 27%. On the other hand, we found a positive correlation between caloric content and the chronic parameters intrinsic rate of natural increase and offspring per female when daphnids were exposed for 120 h to the herbicide.

Heme oxygenase 1 (HO-1) challenges the angiogenic switch in prostate cancer.

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men. Once a tumor is established it may attain further characteristics via mutations or hypoxia, which stimulate new blood vessels. Angiogenesis is a hallmark in the pathogenesis of cancer and inflammatory diseases that may predispose to cancer. Heme oxygenase-1 (HO-1) counteracts oxidative and inflammatory damage and was previously reported to play a key role in prostate carcinogenesis. To gain insight into the anti-tumoral properties of HO-1, we investigated its capability to modulate PCa associated-angiogenesis. In the present study, we identified in PC3 cells a set of inflammatory and pro-angiogenic genes down-regulated in response to HO-1 overexpression, in particular VEGFA, VEGFC, HIF1α and α5ß1 integrin. Our results indicated that HO-1 counteracts oxidative imbalance reducing ROS levels. An in vivo angiogenic assay showed that intradermal inoculation of PC3 cells stable transfected with HO-1 (PC3HO-1) generated tumours less vascularised than controls, with decreased microvessel density and reduced CD34 and MMP9 positive staining. Interestingly, longer term grown PC3HO-1 xenografts displayed reduced neovascularization with the subsequent down-regulation of VEGFR2 expression. Additionally, HO-1 repressed nuclear factor κB (NF-κB)-mediated transcription from an NF-κB responsive luciferase reporter construct, which strongly suggests that HO-1 may regulate angiogenesis through this pathway. Taken together, these data supports a key role of HO-1 as a modulator of the angiogenic switch in prostate carcinogenesis ascertaining it as a logical target for intervention therapy.

Short-term exposure to sublethal tebuconazole induces physiological impairment in male zebrafish (Danio rerio).

The aim of the present study was to assess the physiological response of male zebrafish Danio rerio to the fungicide tebuconazole and recovery in fungicide-free water. Acute toxicity tests were carried out and the median lethal concentration (LC(50)) from 24 to 96 h was calculated. The fish were exposed to a sublethal fungicide concentration of 230 microg/L for 7 or 14 days and allowed to recover for 7 or 14 more days, respectively. Whole-body levels of vitellogenins, triglycerides, cholesterol, glucose, lactate and proteins as well as the activities gamma-glutamil transpeptidase (gamma-GT), alanin aminotransferase (AlAT), alkaline phosphatase (AP) and lactate dehydrogenase (LDH) were assayed; corpulence factor (k) was also calculated. Fish exhibited significant increase of vitellogenins (Vtg), which continued to increase after 14 days of recovery. Levels of glucose, lactate, cholesterol and triglycerides increased after 7 and 14 days of exposure. Finally, cholesterol and glucose recovered after 14 days of recovery whereas triglycerides and lactate continued to be elevated. Proteins and k remained unaltered the entire experiments. AAT, AlAT and AP enhanced during exposure and did not recover at the end (except AlAT). A longer recovery period should be necessary to re-establish fish physiology. These results alert about the multiple disruptive physiological actions that tebuconazole may have on fish.

Biochemical stress response in tetradifon exposed Daphnia magna and its relationship to individual growth and reproduction.

Environmental risk assessment of chemicals toxicity requires the use of costly and labor-intensive chronic data and short-term tests provide additional information. Energy budget is used by the animals for their growth, reproduction, and metabolism and it is reduced in case of toxic stress. Tetradifon acaricide is frequently used in the European Mediterranean region and it is implicated in aquatic environmental pollution. Previous studies showed that the EC(50)-24 h of tetradifon on Daphnia magna was 8.92 mg/L. Based on that, D. magna were exposed to sublethal tetradifon concentrations of 0.10, 0.18, 0.22 and 0.44 mg/L for five days in order to investigate their effect on intermediate metabolism. Caloric content was determined as biomarker of tetradifon toxicity. Results were analyzed using one-way analysis of variance (ANOVA) and Duncan's significant difference test was used to find differences between groups (alpha was set at p=0.05). Daphnids energy content decreased as tetradifon concentration increased. At 120-h caloric content was depleted >51% at pesticide concentrations of 0.18 mg/L and higher. In order to determine a possible link between the 5-d test and the 21-d chronic test, animals under short-term test were exposed to the same pesticide concentrations known to cause adverse effects on reproduction, growth and survival. Results from the present study indicated a good correlation between the proposed 5-day test and daphnid energy budget. Comparison between both, caloric content results and the chronic effect values obtained using life-table studies, suggested a good fit between them. These studies can be used as earlier, predictive and useful tests with comparable results to the classic chronic ones. Our results indicate that caution must be done about the use of tetradifon in the aquatic environment.

Physiological effects of tricyclazole on zebrafish (Danio rerio) and post-exposure recovery.

Short-term effects of tricyclazole on male zebrafish (Danio rerio) physiology were examined joint to the degree of recovery after exposure. Fish were exposed to 142 microg/L (1/100 LC(50)-96 h) of tricyclazole for 7 (Exp.1) and 14 days (Exp.2) and then allowed to recover for 7 or 14 more days, respectively. Whole-body triglycerides, cholesterol, glucose, lactate and total proteins were measured as well as the aspartate aminotransferase (AAT), alanine aminotransferase (AlAT), alkaline phosphatase (AP) and lactate dehydrogenase (LDH) activities as biomarkers of intermediary metabolism; gamma-glutamyl transpeptidase (gammaGT) as biomarker of oxidative detoxification processes and vitellogenin (Vtg) concentration as endpoint for endocrine disruptor effect were also determined. Corpulence factor (k) was calculated. Fungicide exposure in zebrafish resulted in an increased of triglycerides, cholesterol, glucose and lactate levels, however the total protein content did not change. LDH, AlAT and AAT enhanced while AP activity decreased. Corpulence factor (k) also decreased. At the end of the recovery periods cholesterol and glucose levels recovered whereas triglycerides and lactate continued to elevate. Induced disorders on the selected enzymes remained and did not recover at the end of experiments. Fish exhibited significant increases of Vtg during the overall experimental times as a consequence of the fungicide exposure. These findings are of importance in the assessment of the potential risk of new fungicides as tricyclazole on aquatic ecosystems.

Disturbances in energy metabolism of Daphnia magna after exposure to tebuconazole.

This study was conducted to investigate the change of some biochemical parameters in the aquatic invertebrate Daphnia magna following exposure to the fungicide tebuconazole and to determine the most sensitive biomarker among the ones tested in this species. Four biochemical biomarkers (protein, glycogen, lipids and caloric content) were correlated with feeding behaviour studies of D. magna after fungicide exposure. Juveniles of D. magna were exposed to four sublethal concentrations of tebuconazole (0.41, 0.52, 0.71 and 1.14 mgL(-1)) for 5d. Daphnid samples were taken from each test and control group at 24, 48, 72, 96 and 120 h after the start of the experiment. Tebuconazole EC(50) values were calculated on D. magna in our laboratory as 56.83 and 40.10 mgL(-1) at 24 and 48 h, respectively. Results showed that daphnid energy content decreased as tebuconazole concentration increased, especially after 96-120 h of exposure to 0.52 mgL(-1) and higher fungicide concentrations. The data suggest that tebuconazole is moderately toxic to D. magna but also that it seriously impairs the metabolic functions, resulting in alterations in biochemical constituents. In the D. magna feeding study, algae feeding rates were inhibited after fungicide exposure. Such findings indicate the importance of feeding studies in laboratory toxicity test as well as their relationship with others studies. The results emphasize the importance of considering different kind of biomarkers to identify and evaluate the biological effect of a fungicide in the aquatic environment. Although the biochemical biomarkers used resulted good indicators of tebuconazole toxicity, feeding rates in D. magna decreased after only 5h exposure to the fungicide resulting in the most sensitive parameter of daphnid fungicide exposure.

Effects of propanil on the European eel Anguilla anguilla and post-exposure recovery using selected biomarkers as effect criteria.

The aim of this study was to assess the physiological response of Anguilla anguilla to propanil and the degree of recovery after being moved to clean water. Preliminary acute toxicity test was carried out in the laboratory and the median lethal concentration (LC50) at 96 h was calculated as 31.33 mg/L (29.60-33.59 mg/L). NOEC and LOEC values (at 96 h) were also calculated as 20 and 25mg/L, respectively. The fish were exposed to 0.63 and 3.16 mg/L of propanil for 72 h and allowed to recover for 144 h. Total proteins (TPs), gamma-glutamil transpeptidase (gamma-GT), alanin aminotransferase (AlAT), alkaline phosphatase (AP), lactate dehydrogenase (LDH) and water content (WC) were assayed in muscle and liver tissues, liver somatic index (LSI) was also determined. Liver TPs and gamma-GT activity decreased after propanil exposure while AlAT and LDH increased. Muscular AP, AlAT and proteins decreased in intoxicated eels while LDH and gamma-GT activities increased. WC increased in both tissues after herbicide exposure as well as LSI. These results revealed that propanil affects the intermediary metabolism of A. anguilla and that the assayed enzymes can be used as good biomarkers of herbicide contamination. However a longer recovery period should be necessary to re-establish eel physiology. The parameters measured in the present study can be used as herbicide toxicity indicators and are recommended for environmental monitoring assessments.

Direct formulation of a solid foodstuff with phenolic-rich multicomponent solutions from grape seed: effects on composition and antioxidant properties.

The aim of our work was to supplement a solid foodstuff with grape phenolics by osmotic treatment with an aqueous solution made of osmo-active agents (NaCl and sucrose) and a commercial grape seed extract. To investigate how the composition of the osmotic solution affected phenolic infusion, experimental conditions were set by a central composite design with two factors (the molality of NaCl and sucrose in the osmotic solution). In all experiments, the total phenolic content in the osmotic solution was kept constant (6300 +/- 45 mg gallic acid equivalents/kg), and the model food (an agar-agar gel) was processed for 8 h. Throughout the response surface, the osmo-treated model food was significantly supplemented with flavan-3-ols. At the central point of the experimental design, flavan-3-ol monomers and dimers were found in concentrations of 1334 +/- 126 and 486 +/- 55 mg/kg, respectively. Their penetration into the model food was limited by sucrose to a different extent. The Trolox equivalent antioxidant capacity of the osmo-treated gel was higher than that of fruits with a very high free radical scavenging activity.