Sub-chronic exposure of Oreochromis niloticus to environmentally relevant concentrations of smaller microplastics: Accumulation and toxico-physiological responses.

This study assesses the accumulation and toxic effects of environmentally relevant concentrations (0.01, 0.1 and 1 mg/L) of polystyrene MPs (1 µm) in Oreochromis niloticus (Nile tilapia) for 14 days. The results showed that 1 µm PS-MPs accumulated in the intestine, gills, liver, spleen, muscle, gonad and brain. RBC, Hb and HCT showed a significant decline, while WBC and PLT showed a significant increase after the exposure. Glucose, total protein, A/G ratio, SGOT, SGPT and ALP showed significant increments in 0.1 and 1 mg/L of PS-MPs treated groups. The increase in cortisol level and upregulation of HSP70 gene expression in response to MPs exposure indicate MPs-mediated stress in tilapia. MPs-induced oxidative stress is evident from reduced SOD activity, increased MDA levels and upregulated P53 gene expression. The immune response was enhanced by inducing respiratory burst activity, MPO activity and serum TNF-α and IgM levels. MPs exposure also led to down-regulation of CYP1A gene and decreased AChE activity, GNRH and vitellogenin levels, indicating the toxicity of MPs on the cellular detoxification mechanism, nervous and reproductive systems. The present study highlights the tissue accumulation of PS-MP and its effects on hematological, biochemical, immunological and physiological responses in tilapia with low environmentally relevant concentrations.

A Ge.F.I. - ISFG European collaborative study on DNA identification of Cannabis sativa samples using a 13-locus multiplex STR method.

Cannabis sativa is the most used controlled substance in Europe. With the advent of new and less restrictive European laws on cannabis sale for recreational use (including in Italy), an increase in indoor cannabis crops were observed. This increase was possible due to the availability of cannabis seeds through the internet market. Genetic identification of cannabis can link seizures and if in possession then might aid in an investigation. A 13-locus multiplex STR method was previously developed and validated by Houston et al. A collaborative exercise was organized by the Italian Forensic Geneticists - International Society of Forensic Genetics (Ge.F.I. - ISFG) Working Group with the aim to test the reproducibility, reliability and robustness of this multiplex cannabis STR kit. Twenty-one laboratories from three European countries participated in the collaborative exercise and were asked to perform STR typing of two cannabis samples. Cannabis DNA samples and the multiplex STR kit were provided by the University of Barcelona and Sam Houston State University. Different platforms for PCR amplification, capillary electrophoresis (CE) and genotyping software were selected at the discretion of the participating laboratories. Although the participating laboratories used different PCR equipment, CE platforms and genotyping software, concordant results were obtained from the majority of the samples. The overall genotyping success ratio was 96%. Only minor artifacts were observed. The mean peak height ratio was estimated to be 76.3% and 78.1% for sample 1 and sample 2, respectively. The lowest amount of -1 / + 1 stutter percentage produced, when the height of the parent allele was higher than 8000 RFU, resulted to be less than 10% of the parent allele height. Few common issues were observed such as a minor peak imbalance in some heterozygous loci, some artifact peaks and few instances of allelic drop-out. The results of this collaborative exercise demonstrated the robustness and applicability of the 13-locus system for cannabis DNA profiling for forensic purposes.

Outbreaks of epizootic ulcerative syndrome in Kerala, India following episodes of flooding.

Large-scale fish mortality was observed in flood-affected fish farms across several parts of Kerala following heavy rainfall in August 2018 and 2019-nearly 53% above the normal monsoon rain that the region receives. The affected fish had severe haemorrhages and ulcers, typical of the highly infectious disease epizootic ulcerative syndrome (EUS) caused by the water mould Aphanomyces invadans. In freshwater, snakeheads Channa spp. and in brackish water mullet (Mugilidae) and pearl spot (Etroplus suratensis) were severely affected. EUS was observed in 4 freshwater fishes for the first time: dotted sawfin barb Pethia punctata (Cyprinidae), Malabar leaffish Pristolepis malabarica (Pristolepididae), mahecola barb Puntius mahecola (Cyprinidae) and giant snakehead Channa pseudomarulius (Channidae). Histology and molecular diagnosis confirmed the cause of mortality to be EUS. Fungal hyphae were also observed in deeply ulcerated fish, revealed by lactophenol cotton blue staining. The severity of the EUS outbreak was linked to the sudden change in water quality associated with the flood, such as lower water temperature, and decreases in pH, total alkalinity and total hardness.

Probabilistic cost-effectiveness analysis of controlled ovarian stimulation with recombinant FSH plus recombinant LH vs. human menopausal gonadotropin for women undergoing IVF.

BACKGROUND: The association of recombinant FSH plus recombinant LH in 2:1 ratio may be used not only to induce ovulation in anovulatory women with hypogonadotropic hypogonadism but also to achieve multiple follicular developments in human IVF. The aim of this analysis was to estimate the cost-effectiveness of Controlled Ovarian Stimulation (COS) with recombinant FSH (rFSH) plus recombinant LH (rLH) in comparison with highly purified human menopausal gonadotropin (HP-hMG) in the woman undergoing in vitro fertilization (IVF) in Italy. METHODS: A probabilistic decision tree was developed to simulate patients undergoing IVF, either using r-FSH + r-LH or HP-hMG to obtain COS. The model considers the National Health System (NHS) perspective and a time horizon equal to two years. Simulations were reported considering the number of retrieved oocytes (5-9, 10-15 and > 15) and transition probabilities were estimated through specific analyses carried out on the population of 848 women enrolled in the real-life. RESULTS: The model estimated that patients undertaking therapeutic protocol with r-FSH + r-LH increase the general success rate (+ 6.6% for pregnancy). The incremental cost-effectiveness ratio (ICER) per quality-adjusted life year (QALY) of r-FSH + r-LH was below the willingness to pay set at €20,000 for all the considered scenarios. CONCLUSIONS: The cost-utility analysis demonstrated that the r-FSH + r-LH is a cost-effective option for the Italian National Health System (NHS).

Time-dependent changes and prognostic value of lactatemia during the first 24 h of life in brachycephalic newborn dogs.

Blood lactate concentration is known to be a good prognostic indicator associated with the severity of illness and the patient's outcome both in human and veterinary medicine. It also plays a significant role in the assessment of the newborn, being a good indicator of fetal hypoxia and the ideal predictor of morbidity at term in babies. In veterinary neonatal medicine, hyperlactatemia is considered a valid prognostic marker in critically ill foals; moreover, blood lactate measurement has been proposed for the evaluation of newborn viability and the assessment of fetal distress during delivery in dogs. Unfortunately, only a few studies have been published concerning the canine species. The present work examines 67 brachycephalic newborn dogs and their mothers, with the aim to evaluate the time-dependent changes of blood lactate and glucose concentration during the first 24 h after vaginal or caesarean delivery both in puppies and bitches. To our knowledge, this is the first published study examining the time-dependent changes of these parameters in the bitch after parturition. Within the studied population of puppies, non-surviving was significantly associated with a higher lactatemia and a lower APGAR score. Blood lactate was high at birth then progressively decreased during the first 24 h of life and a lack of normalization of blood lactate levels within this time interval was suggestive for a poor prognosis for the newborn dogs; moreover, the decrease appeared to be slower after vaginal delivery. Lactatemia also showed a positive correlation with glycemia at birth. Concerning the bitches examined, blood lactate was found to be significantly higher after vaginal delivery than after caesarean section; the normalization occurred within 24 h after parturition. Blood glucose level was significantly higher at 2 h from delivery both in the group of bitches submitted to caesarean section and in those undergoing natural whelping but no statistical correlation was found between maternal glycemia and lactatemia. The results of the present study highlighted that the monitoring of lactatemia during the first 24 h of life, in association with the assessment of the APGAR score at birth, can be an useful prognostic tool helping to identify the most severely distressed puppies and to provide them an adequate support.

Influence of Different Protocols of Urethral Catheterization after Pharmacological Induction (Ur.Ca.P.I.) on Semen Quality in the Domestic Cat.

The aim of this study was to evaluate the influence that different protocols of urethral catheterization after pharmacological induction (Ur.Ca.P.I.) may have on the semen quality of the domestic cat. The study has been divided into two experiments: one in which different dosages of medetomidine administrated are evaluated and the second one in which the timing of the catheterization after pharmacological induction is tested. In the first experiment, 18 cats were sedated with the recommended dosage of medetomidine (130 µg/kg i.m.) while the other 18 were sedated with a lower dose of the same drug (50 µg/kg i.m.). In the second experiment, three groups were implemented, each containing 25 subjects. In group 1, the semen collection was performed immediately once the pharmacological effect of the drug was reached; in group 2, the semen collection was performed three times every 5 min after the pharmacological effect was reached; finally, in group 3, Ur.Ca.P.I. was performed 20 min after the pharmacological effect was reached. All the different protocols permitted sperm collection, nevertheless the first experiment showed a better quality in terms of volume, concentration, total number of spermatozoa (p < 0.01) and quality of the movement (motility p < 0.05 and forward progressive motility p < 0.01), using a high medetomidine dosage rather than 50 µg/kg i.m. In the second experiment, forward motility was statistically higher (p < 0.01) in the first group and total volume was higher (p < 0.01) in the second and third group, while other parameters were statistically not different. Results suggest that a single catheterization immediately after the onset of the pharmacological effect leads to a good-quality semen with the lowest possibility of damaging the urethra and that a sedation with 130 µg/kg of medetomidine leads to a better quality sperm collection than 50 µg/kg does.

Development of an Italian RM Y-STR haplotype database: Results of the 2013 GEFI collaborative exercise.

Recently introduced rapidly mutating Y-chromosomal short tandem repeat (RM Y-STR) loci, displaying a multiple-fold higher mutation rate relative to any other Y-STRs, including those conventionally used in forensic casework, have been demonstrated to improve the resolution of male lineage differentiation and to allow male relative separation usually impossible with standard Y-STRs. However, large and geographically-detailed frequency haplotype databases are required to estimate the statistical weight of RM Y-STR haplotype matches if observed in forensic casework. With this in mind, the Italian Working Group (GEFI) of the International Society for Forensic Genetics launched a collaborative exercise aimed at generating an Italian quality controlled forensic RM Y-STR haplotype database. Overall 1509 male individuals from 13 regional populations covering northern, central and southern areas of the Italian peninsula plus Sicily were collected, including both "rural" and "urban" samples classified according to population density in the sampling area. A subset of individuals was additionally genotyped for Y-STR loci included in the Yfiler and PowerPlex Y23 (PPY23) systems (75% and 62%, respectively), allowing the comparison of RM and conventional Y-STRs. Considering the whole set of 13 RM Y-STRs, 1501 unique haplotypes were observed among the 1509 sampled Italian men with a haplotype diversity of 0.999996, largely superior to Yfiler and PPY23 with 0.999914 and 0.999950, respectively. AMOVA indicated that 99.996% of the haplotype variation was within populations, confirming that genetic-geographic structure is almost undetected by RM Y-STRs. Haplotype sharing among regional Italian populations was not observed at all with the complete set of 13 RM Y-STRs. Haplotype sharing within Italian populations was very rare (0.27% non-unique haplotypes), and lower in urban (0.22%) than rural (0.29%) areas. Additionally, 422 father-son pairs were investigated, and 20.1% of them could be discriminated by the whole set of 13 RM Y-STRs, which was very close to the theoretically expected estimate of 19.5% given the mutation rates of the markers used. Results obtained from a high-coverage Italian haplotype dataset confirm on the regional scale the exceptional ability of RM Y-STRs to resolve male lineages previously observed globally, and attest the unsurpassed value of RM Y-STRs for male-relative differentiation purposes.

Endoscopic transcervical catheterization in the domestic cat.

Feline-assisted reproduction is still not routinely performed in veterinary practice, although there is an increasing interest on the subject by cat breeders. In recent years, many techniques for artificial insemination in the domestic cat have been developed with regard to the intrauterine deposition of sperm through the catheterization of the cervix. Transcervical catheterization has been described also for diagnostic and therapeutic purposes. This article provides the first description of a new method for cervical catheterization, under the direct visualization of the cervix, using a rigid endoscope and a new specially designed transcervical catheter. The procedure was performed on 14 queens with a success rate of 85.71%.

In vitro gene and chromosome characterization of expanded bone marrow mesenchymal stem cells for musculo-skeletal applications.

OBJECTIVE: A number of studies have shown the role of expanded Bone Marrow-derived Mesenchymal Stem Cells in the repair and regeneration of musculo-skeletal tissues. The current European regulations define in vitro expanded cells for clinical purposes as substantially manipulated and include them in the class of Advanced-Therapy Medicinal Products to be manufactured in compliance with current Good Manufacturing Practice. Among the characteristics that such cells should display, genomic stability has recently become a major safety concern. The aim of this study is to perform a chromosomal and genetic characterization of Bone Marrow-derived Mesenchymal Stem Cells expanded in compliance with Good Manufacturing Practice for a potential clinical use in orthopaedics. MATERIALS AND METHODS: Mesenchymal Stem Cells, isolated from bone marrow, were expanded for six weeks in compliance with current Good Manufacturing Practice. DNA profiling analyses were applied to test cross-contamination absence. Genomic stability was evaluated by means of karyotyping, sequencing of TP53, p21/CDKN1A and MDM2 genes and the expression analysis of c-MYC and H-RAS oncogenes, p21/CDKN1A, TP53, p16/CDKN2A, RB1 and p27/CDKN1B tumor suppressor genes and hTERT gene. RESULTS: The DNA profiling analysis showed a unique genetic profile for each Mesenchymal Stem Cell culture, indicating the absence of cross-contamination. Karyotyping evidentiated some chromosomal abnormalities within the 10% limit set by the Cell Products Working Party review, except for one patient. In all cases, the molecular biology analyses did not revealed DNA point mutations, acquisition or changes in gene expression. hTERT levels were undetectable. CONCLUSIONS: Cultured Mesenchymal Stem Cells do not seem to be prone to malignant transformation. In fact, although some chromosomal aberrations were found, molecular biology analyses demonstrated that the expansion phase did not induce the acquisition of de novo genetic changes.

A novel DNA profiling application for the monitoring of cross-contamination in autologous chondrocyte implantation.

BACKGROUND: Autologous chondrocyte implantation (ACI) is a cell-based treatment that can be used to regenerate chondral defects. European legislation specifically classifies such produced chondrocytes as "medicinal for advanced cell therapy" that have to be manufactured in pharmaceutical factories according to specific rules, named Good Manufacturing Practices (GMPs). One main requirement of cell manipulation in advanced therapy is to prevent the risk of any contamination. AIM: The aim of this study was to verify if chondrocyte cultures suitable for ACI were free of cross-contamination by means of DNA profiling techniques. MATERIALS AND METHODS: Cell cultures were carried on in a Hospital Cell Factory in compliance with European current Good Manufacturing Practices. DNA profiling, by means of Short Tandem Repeats and miniShort Tandem Repeats analyses, was performed on expanded chondrocytes and their related control blood samples. Mitochondrial DNA was analysed to further confirm the results and to evaluate possible mutations occurred in the samples. RESULTS: Our findings demonstrated the absence of cross-contamination between chondrocyte cultures and, thus, their identity maintenance until the end of the manipulation. CONCLUSIONS: DNA profiling technique can be a suitable test for quality control not only for chondrocyte manipulation, but for cell therapy in general.

Carbon stock evaluation from topsoil of forest stands in NE Italy.

Gas emissions from anthropic activities, particularly CO2, are responsible for global warming. Soil is a major carbon sink on a planetary level, thereby contributing to mitigate greenhouse effect. In the present work, the objectives were: 1) to evaluate the topsoil carbon stock of different forest stands in NE Italy, and 2) to outline the relationships among humus forms, soil organic matter dynamics, and actual carbon stock under different vegetation coverage, with reference to climate change. Five forest stands and the related topsoils, were selected in the Dolomites area. The humus forms were examined in the field and samples were carried to the lab for further physical-chemical analyses. The carbon stock for each soil was calculated by means of pedotransfer functions. The less developed humus forms, as the Dysmull and the Hemimoder, presented the highest carbon storage capacity (168 t/y and 129 t/y), followed by Lithoamphimus (123 t/y) and Eu-amphimus (96 t/y), and by Oligomull (86 t/y). Organic horizons proved to recover 36% of the total carbon stocked along the soil profile, and this points to humus layers as a fundamental tool in carbon stock evaluation. Positive correlations between elevation, humus forms and soil carbon pools were found.

[Evaluation of the impact of night-work on health in a population of workers in Tuscany]. / Valutazione dell'impatto del lavoro notturno sulla salute in una popolazione di lavoratori della Toscana.

Night-work, loading to subversion of physiological circadian rhythms and habits, may cause a lowering of work performance, an increase in the risk of accidental events and, more generally, a perception of less satisfactory living condition. In our experience we have interviewed 359 workers (night-workers and not). We found, in line with the Literature, that night-workers receive a less satisfactory quality of sleep, often associated with daytime sleepiness. The prevalence of disorders of various organs and systems is higher in these subjects, as well as the consumption of caffeine and nicotine. Regarding road and work-accidents, a similar trend is not so clearly confirmed in our series; in this context we have, nevertheless, to take account of some potential bias. Our experience confirmed that night-work may produce animpact on the individual overall well-being and, consequently, on the companies' productivity. To reduce this impact and the related costs, it is necessary to plan interventions on both workers and work organization.

mtDNA HVI length heteroplasmic profile in different tissues of maternally related members.

Sequencing of the homopolymeric tract of cytosines (C-stretch) in human mitochondrial HVI region usually results in a blurred pattern beyond it when a T/C transition at nt 16189 occurs: it depends on a length heteroplasmy probably arising through a replication slippage. This study aims to investigate the distribution of heteroplasmic length variants within three related individuals along maternal lineage by cloning approach. Sequencing of multiple independent clones (12--14) is sufficient to yield heteroplasmic profiles. In addition, we illustrate a direct correlation between expansion of heteroplasmy modal length and reduction of the number of adenines preceding the homopolymeric tract; this association may be useful in pedigree analysis and in forensic field for tissues comparison, single hair sample included.

Sex chromosome analysis in Turner Syndrome by a pentaplex PCR assay.

In this study, we describe a pentaplex PCR to determine the parental origin of the X chromosome and the presence of mosaicism, via amplification of four polymorphic markers located along the X chromosome (DXS10011, DXS6807, HUMARA, DXS101) and the X-Y amelogenin marker, in 41 families having a daughter with Turner Syndrome. Our results confirmed the cytogenetic findings and we found that the parental origin of the single X chromosome to be maternal in 84% of cases.

Different informativeness of the three hypervariable mitochondrial DNA regions in the population of Bologna (Italy).

Mitochondrial DNA (mtDNA) sequence variations at hypervariable regions HVI, HVII and HVIII were analysed in 100 unrelated Italians from Bologna. The values of the statistical parameters are in agreement with the range of European populations. We suggest that the less informative HVIII region may be useful to distinguish HVI-HVII identical sequences in forensic analysis especially when nuclear DNA cannot be investigated.

Automated fluorescence analysis of CAG repeats at the human androgen receptor gene (HUMARA): evaluation of polymorphism in an Italian sample and report of a new allele.

The HUMARA CAG repeats polymorphism was studied in an Italian population sample. Polymerase chain reaction amplification and automated fluorescent analysis were used. A total of 19 and 15 repeats was observed in female and male subjects, respectively, and one new allele was found. The authors conclude that this X-linked short tandem repeat, typed without ambiguity and with a heterozygosity of 0.902, is useful in parentage testing of female subjects.