Molecular analysis of endocrine disruption in hornyhead turbot at wastewater outfalls in southern california using a second generation multi-species microarray.

Sentinel fish hornyhead turbot (Pleuronichthysverticalis) captured near wastewater outfalls are used for monitoring exposure to industrial and agricultural chemicals of ~ 20 million people living in coastal Southern California. Although analyses of hormones in blood and organ morphology and histology are useful for assessing contaminant exposure, there is a need for quantitative and sensitive molecular measurements, since contaminants of emerging concern are known to produce subtle effects. We developed a second generation multi-species microarray with expanded content and sensitivity to investigate endocrine disruption in turbot captured near wastewater outfalls in San Diego, Orange County and Los Angeles California. Analysis of expression of genes involved in hormone [e.g., estrogen, androgen, thyroid] responses and xenobiotic metabolism in turbot livers was correlated with a series of phenotypic end points. Molecular analyses of turbot livers uncovered altered expression of vitellogenin and zona pellucida protein, indicating exposure to one or more estrogenic chemicals, as well as, alterations in cytochrome P450 (CYP) 1A, CYP3A and glutathione S-transferase-α indicating induction of the detoxification response. Molecular responses indicative of exposure to endocrine disruptors were observed in field-caught hornyhead turbot captured in Southern California demonstrating the utility of molecular methods for monitoring environmental chemicals in wastewater outfalls. Moreover, this approach can be adapted to monitor other sites for contaminants of emerging concern in other fish species for which there are few available gene sequences

Molecular tools for contaminated sediment monitoring

La presente tesi di dottorato ha posto i primi passi con le domande generate dalle ricerche precedenti e considerando i problemi esistenti per le contaminazione ambientali. Il presente progetto è stato effettuata in tre fasi. In primo luogo, abbiamo verificato l'idoneità di Solea solea come bioindicatore per valutare l'effetto biologico di sedimenti contaminati ambientali sugli organismi acquatici. Una serie di geni sono stati identificati mediante DNA microarray e alcuni di essi sono stati parzialmente clonati e ottimizzati per il loro uso come biomarker di tossicità. Questi geni sono stati quantificati mediante Real Time PCR. Abbiamo dimostrato che la caratterizzazione chimico / fisiche di sedimenti contaminati non sono sufficienti per determinare la tossicità dei sedimenti (capitolo 1). Successivamente, abbiamo analizzato il pool di geni ottimizzati in Sparus aurata per valutare la differenza dell’effetto biologico di due sedimenti contaminati (campionati nello stesso luogo, il Nord Est d'Italia). Possiamo concludere che tutti i biomarcatori ed il bioindicatore qui utilizzati hanno un’alta capacità di rispondere a bassa tossicità di sedimenti contaminati che in questo caso ha effetti più nocivi sugli organismi animali più elevati rispetto a quelli contaminati (capitolo 2), infine, si analizza l'uso di innovative tecniche di biologia molecolare (microarray), nel monitoraggio ambientale delle costiera della California meridionale. Questi risultati evidenziano l'utilità di un approccio multi-specie microarray come strumento diagnostico sensibile per la presenza di interferenti endocrini in ambiente acquatico (capitolo 3). L'integrazione di innovative tecniche molecolari per il monitoraggio ambientale prodotto risultati che contribuiscono in maniera significativa alla valutazione della marina sedimenti contaminati e al miglioramento della gestione del pollution.and ambiente marino Inoltre, questo studio rivela nuove possibilità di ricerca per individuare nuovi bioindicatore europeo Ecosistema e nuovi biomarcatori per lo sviluppo di un responsabile di linee guida di gestione dei sedimenti contaminati

Inclusion of small intestinal absorption and simulated mucosal surfaces further improve the Mucosal Simulator of the Canine Intestinal Microbial Ecosystem (M-SCIME™)

While a large set of in vitro models are available to study the effects of specific food ingredients (e.g. pre- and probiotics) on the human gut microbiome, the availability of such models for companion animals is limited. Since improving gut health of such animals is an emerging research field, the Simulator of the Canine Intestinal Microbial Ecosystem (SCIME™) was recently developed and validated with in vivo data. The current study presents a further improvement of this model by using an alternative method for feed preparation, i.e. by administering digestive enzymes to mimic upper gastro-intestinal digestion followed by a dialysis approach to mimic small intestinal absorption. As opposed to the previously implemented method, this resulted in a more optimal simulation of protein digestion and absorption. Further, upon entrance in the colon, increased production of the health-promoting butyrate and lower levels of Lactobacillus spp. and Bifidobacterium spp. were observed, which corresponded better with obtained in vivo data. A second model improvement consisted of the implementation of a mucosal environment to not only simulate luminal but also mucosal microbiota. In consistency with the human model for which this technology was previously validated, it was found that for all canine microbiota mucin beads were enriched with members of the Lachnospiraceae (~ Clostridium cluster XIVa), a family containing multiple well-known butyrate producers. The SCIME™ was thus upgraded to a so-called Mucosal SCIME™ (M-SCIME™). In conclusion, the current study presents improvements of the SCIME™, further increasing the relevance of obtained data with this in vitro model for dogs

Yeast-derived formulations are differentially fermented by the canine and feline microbiome as assessed in a novel in vitro colonic fermentation model

The current study evaluated the effect of five yeast-derived formulations (T1-T5) on microbial metabolism and composition of the canine and feline gut microbiota using a novel in vitro colonic incubation approach. This novel in vitro model allowed for growth of the entire spectrum of dog- and cat-derived bacteria from the inoculum, thus offering an excellent platform to evaluate effects of nutritional interventions on the gut microbiota. Further, yeast-derived ingredients differentially increased production of acetate, propionate, butyrate, ammonium, and branched short-chain fatty acids, with TS and T1 consistently stimulating propionate and butyrate, respectively. 16S-targeted Illumina sequencing coupled with flow cytometry provided unprecedented high-resolution quantitative insights in canine and feline microbiota modulation by yeast-derived ingredients, revealing that effects on propionate production were related to Prevotellaceae, Tannerellaceae, Bacteroidaceae, and Veillonellaceae members, while effects on butyrate production were related to Erysipelotrichaceae, Lachnospiraceae, Ruminococcaceae, and Fusobacteriaceae. Overall, these findings strengthen the health-promoting potential of yeast-derived ingredients

Complementary feed for the control of pruritus in atopic dermatitis in dogs

Pruritus is a common manifestation in dogs with allergic skin diseases and itching can significantly affect the quality of life of both affected animals and their owners, with even severe repercussions [1]. Pharmacological treatments and complementary feeds that are able to control itching quickly and in the long run are in great demand and attract the attention of many researchers and companies. The aim of this study was to assess the effectiveness of a complementary feed containing flavonoids, stilbenes, and cannabinoids (obtained from vegetable/botanical by- products/vegetable/botanical source) in the control of itching in dogs suffering from atopic dermatitis. Such complementary feed has been shown to be able to reduce the gene expression of ccl2, ccl17, il31ra and tslp in an experimental in vitro model of atopic dermatitis [3]. The primary efficacy endpoint was the reduction of CADESI-04 and pruritus visual analogue scale (pVas) scores. The study protocol was successfully submitted to the Animal Welfare Body of the University of Camerino (protocol code 10/2021). Ten dogs affected by atopic dermatitis, diagnosed according to current guidelines [1, 2], received a hypoallergenic food for the duration of the study. Once enrolled, in the first 6 weeks dogs received the administration of oclacitinib (Apoquel®, Zoetis) twice daily for two weeks and then once daily for 4 weeks. Starting from the fifth week, the administration of complementary feed began, according to the following dosage: twice daily for two weeks and then once daily for 8 weeks. Administration of oclacitinib was discontinued at week 6 in all dogs enrolled in the study, who received the complementary feed up to week 12. In all dogs there was a marked reduction in both CADESI-04 and owner-reported pVas for pruritus in the first four weeks of oclacitinib administration. In the fifth and sixth week of the study (oclacitinib + complementary feed) the trend of CADESI-04 and pVas was the same, as well as from the seventh week onwards for all dogs enrolled in the study. Although data collected are only preliminary, it is possible to highlight that the complementary feed effectively control itching in supplemented dogs, which did not show any adverse event. This study further confirm the ability of selected complementary feed to control dermatological disease manifestation in dogs [4]. COMPLEMENTARY FEED FOR THE CONTROL OF PRURITUS IN ATOPIC DERMATITIS IN DOGS Andrea Marchegiani (1), Alessandro Fruganti (1), Elena Dalle Vedove (2), Benedetta Bachetti (2), Marcella Massimini (2), Cataldo Ribecco (2), Matteo Cerquetella (1), AndreaSpaterna (1) (1) Università degli Studi di Camerino, Scuola di Bioscienze e Medicina Veterinaria. (2) Research and Development Unit (NIL), C.I.A.M. srl. Corresponding author: A. Marchegiani ([email protected]) [1] Favrot C et al. A prospective study on the clinical features of chronic canine atopic dermatitis and its diagnosis. Vet Dermatol, 21:23–31, 2010. [2] Hensel P et al. Canine atopic dermatitis: detailed guidelines for diagnosis and allergen identification. BMC Vet Res, 11(1):196, 2015. [3] Massimini M et al. Polyphenols and Cannabidiol Modulate Transcriptional Regulation of Th1/Th 2 Inflammatory Genes Related to Canine Atopic Dermatitis. Front Vet Sci, 8:1–14, 2021. [ 4] M arc hegi ani A et al . Im pac t o f N utri ti o na l Supplementation on Canine Dermatological Disorders. Vet Sci MDPI, 38:1–13, 2020

Molecular analysis of endocrine disruption in hornyhead turbot at wastewater outfalls in southern california using a second generation multi-species microarray.

Sentinel fish hornyhead turbot (Pleuronichthysverticalis) captured near wastewater outfalls are used for monitoring exposure to industrial and agricultural chemicals of ~ 20 million people living in coastal Southern California. Although analyses of hormones in blood and organ morphology and histology are useful for assessing contaminant exposure, there is a need for quantitative and sensitive molecular measurements, since contaminants of emerging concern are known to produce subtle effects. We developed a second generation multi-species microarray with expanded content and sensitivity to investigate endocrine disruption in turbot captured near wastewater outfalls in San Diego, Orange County and Los Angeles California. Analysis of expression of genes involved in hormone [e.g., estrogen, androgen, thyroid] responses and xenobiotic metabolism in turbot livers was correlated with a series of phenotypic end points. Molecular analyses of turbot livers uncovered altered expression of vitellogenin and zona pellucida protein, indicating exposure to one or more estrogenic chemicals, as well as, alterations in cytochrome P450 (CYP) 1A, CYP3A and glutathione S-transferase-α indicating induction of the detoxification response. Molecular responses indicative of exposure to endocrine disruptors were observed in field-caught hornyhead turbot captured in Southern California demonstrating the utility of molecular methods for monitoring environmental chemicals in wastewater outfalls. Moreover, this approach can be adapted to monitor other sites for contaminants of emerging concern in other fish species for which there are few available gene sequences

Q-PCR validation of Multi-Species Endocrine Microarray.

<p>Multi-species SYBR green Q-PCR validation of multi-species microarray for (A) <i>CYP3A</i> (B) <i>Vit1</i> (C) <i>Vit2</i> (D) ESR1/<i>ERα</i> (E) ESR2/<i>ERβ</i> specific transcripts. <i>GAPDH</i>-normalized fold changes (based on triplicate measurements) of gene expression in turbot liver from selected impacted sites with respect to reference fish are presented. Each fold change was derived from the mean log₂ ratio between each fish and a reference derived from two control fish. <i>Vit1</i> and <i>Vit2</i> transcripts were strongly up-regulated in all fish. <i>ERα</i> was down-regulated in one fish and up-regulated in two others relative to control fish. <i>ERβ</i> was down-regulated in all fish examined relative to control fish. <i>CYP3A</i> was up-regulated in eight and down-regulated in two fish.</p

Quality control specifications for the second generation multi-species endocrine microarray.

<p>Comparison of the performance characteristics of on both prototype (spotted oligonucleotide) and second generation (Agilent ink jet oligonucleotide) endocrine multi-species microarray platforms; Panels A and B: scatter plots of log₂ signal intensities of an individual fish from the Los Angeles Sanitation District versus the control fish pool. Panels A and B depict prototype and second generation platforms respectively. Panel C; direct comparison of platform sensitivity defined as log₂ ratio between treatment and ABC control. Panel D: Analysis of signal concordance. Panel E: Analysis of variance with both platforms. To measure internal consistency (variance of internal replicates), log₂ transformed expression value was plotted on the x-axis. Standard deviation is plotted on the y-axis, with a range of plus or minus one standard deviation from the mean expression value. The y-axis is a measure of the deviation of individual replicate probes stray from the mean value. The second generation platform has smaller variance of the internal replicates. Probe variance on both platforms is independent of the signal. Panel F: Comparison of the probe signal intensity distribution (plotted as log₂ transformed) for 149 probes that were present on both platforms.</p