Transcriptomic approaches to study the effects of xenobiotics in ruminants

This thesis is concerned with the study and characterization of the xenobiotics-induced transcriptomic signature in some ruminants. Based on the different studies presented in this thesis, the microarray-based transcriptomics approach was able to provide a holistic view on the global gene expression in diverse types of tissues – namely, skeletal muscle, liver, whole blood, primary hepatocytes- and kidney-derived cell lines. The pre-designed commercial bovine microarray enabled the discovery of many biomarkers with which the differentiation between illicitly-treated and untreated veal calves was possible. It also demonstrated the transcriptomic signature dissimilarity between 2 tissues (i.e. skeletal muscle and liver) exposed to the same treatment (i.e. anabolic steroids). Also, the same approach revealed the presence of some transcriptomic landscape convergence between the hepatocytes primary cultures and the Madin-Darby bovine kidney (MDBK) cell line, which in turn spots the light on the MDBK cells as a possible surrogate in vitro tool for some liver-based functional studies. Finally, a custom-designed whole-transcriptome sheep (Ovis aries) microarray revealed the immune-system-induction and the transcriptional-modulation capacity of organic selenium in sheep. Collectively, the transcriptomics approach overcame the shortcoming of focusing on changes in expression of a priori list of selected genes – instead, it looks at the bigger picture within the protein-coding part of the genome. It is important to mention that using an alternative functional analysis tools [i.e. Gene set enrichment analysis (GSEA)] was useful to cross-validate the output of the conventional overrepresentation tools like the Database for Annotation, Visualization and Integrated Discovery (DAVID). The collective body of work represented here shows the adequacy of using microarray, commercial and custom-designed, to depict a holistic picture about the global gene expression profile of a given tissue. Still, there are some challenges in data analysis, interpretation and integration with the output of other alternative omic techniques – those challenges are highlighted and discussed across the different chapters of this thesis

RNA sequencing-based whole-transcriptome analysis of friesian cattle fed with grape pomace-supplemented diet

Grape pomace (GPO), the main by-product of the wine making process, is a rich source of polyphenols with potent antioxidant properties. Recently, GPO has emerged as a potential feed additive in livestock nutrition, with several reports describing its beneficial effects on animals’ overall health status or production traits. However, little is known about it from a molecular biology standpoint. In the present study, we report the first RNA sequencing-based whole-transcriptome profiling of Friesian calves fed with a GPO-supplemented diet. We identified 367 differentially expressed genes (p < 0.05) in the GPO-supplemented calves (n = 5), when compared with unsupplemented control group (n = 5). The pathway analysis showed that ‘cholesterol lipid biosynthesis’ was the most negatively-enriched (p < 0.001) pathway in the GPO-supplemented animals. In specific terms, five important genes coding for cholesterol biosynthesis enzymes, namely the Farnesyl-diphosphate Farnesyltransferase 1 (FDFT-1), Squalene Epoxidase (SQLE), NAD(P)-dependent Steroid Dehydrogenase-like (NSDHL), Methylsterol Monooxygenase (MSMO)-1, and Sterol-C5-desaturase (SC5D), two major transcription factors (the Sterol Regulatory Element-binding Transcription Factor 1 and 2), as well as the Low-Density Lipoprotein Receptor (LDLR), were all downregulated following GPO supplementation. Such an effect was mirrored by a reduction of blood cholesterol levels (p = 0.07) and a lowered (p < 0.001) Malondialdehyde (lipid oxidation marker) level in carcasses. We provide evidence on the effects of GPO-supplemented diets on the whole-transcriptome signature in veal calves, which mainly reflects an antioxidant activity

Modeling glioblastoma heterogeneity as a dynamic network of cell states

Tumor cell heterogeneity is a crucial characteristic of malignant brain tumors and underpins phenomena such as therapy resistance and tumor recurrence. Advances in single-cell analysis have enabled the delineation of distinct cellular states of brain tumor cells, but the time-dependent changes in such states remain poorly understood. Here, we construct quantitative models of the time-dependent transcriptional variation of patient-derived glioblastoma (GBM) cells. We build the models by sampling and profiling barcoded GBM cells and their progeny over the course of 3\ua0weeks and by fitting a mathematical model to estimate changes in GBM cell states and their growth rates. Our model suggests a hierarchical yet plastic organization of GBM, where the rates and patterns of cell state switching are partly patient-specific. Therapeutic interventions produce complex dynamic effects, including inhibition of specific states and altered differentiation. Our method provides a general strategy to uncover time-dependent changes in cancer cells and offers a way to evaluate and predict how therapy affects cell state composition

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10&nbsp;years; 78.2% included were male with a median age of 37&nbsp;years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Global, regional, and national burden of disorders affecting the nervous system, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BackgroundDisorders affecting the nervous system are diverse and include neurodevelopmental disorders, late-life neurodegeneration, and newly emergent conditions, such as cognitive impairment following COVID-19. Previous publications from the Global Burden of Disease, Injuries, and Risk Factor Study estimated the burden of 15 neurological conditions in 2015 and 2016, but these analyses did not include neurodevelopmental disorders, as defined by the International Classification of Diseases (ICD)-11, or a subset of cases of congenital, neonatal, and infectious conditions that cause neurological damage. Here, we estimate nervous system health loss caused by 37 unique conditions and their associated risk factors globally, regionally, and nationally from 1990 to 2021.MethodsWe estimated mortality, prevalence, years lived with disability (YLDs), years of life lost (YLLs), and disability-adjusted life-years (DALYs), with corresponding 95% uncertainty intervals (UIs), by age and sex in 204 countries and territories, from 1990 to 2021. We included morbidity and deaths due to neurological conditions, for which health loss is directly due to damage to the CNS or peripheral nervous system. We also isolated neurological health loss from conditions for which nervous system morbidity is a consequence, but not the primary feature, including a subset of congenital conditions (ie, chromosomal anomalies and congenital birth defects), neonatal conditions (ie, jaundice, preterm birth, and sepsis), infectious diseases (ie, COVID-19, cystic echinococcosis, malaria, syphilis, and Zika virus disease), and diabetic neuropathy. By conducting a sequela-level analysis of the health outcomes for these conditions, only cases where nervous system damage occurred were included, and YLDs were recalculated to isolate the non-fatal burden directly attributable to nervous system health loss. A comorbidity correction was used to calculate total prevalence of all conditions that affect the nervous system combined.FindingsGlobally, the 37 conditions affecting the nervous system were collectively ranked as the leading group cause of DALYs in 2021 (443 million, 95% UI 378–521), affecting 3·40 billion (3·20–3·62) individuals (43·1%, 40·5–45·9 of the global population); global DALY counts attributed to these conditions increased by 18·2% (8·7–26·7) between 1990 and 2021. Age-standardised rates of deaths per 100 000 people attributed to these conditions decreased from 1990 to 2021 by 33·6% (27·6–38·8), and age-standardised rates of DALYs attributed to these conditions decreased by 27·0% (21·5–32·4). Age-standardised prevalence was almost stable, with a change of 1·5% (0·7–2·4). The ten conditions with the highest age-standardised DALYs in 2021 were stroke, neonatal encephalopathy, migraine, Alzheimer's disease and other dementias, diabetic neuropathy, meningitis, epilepsy, neurological complications due to preterm birth, autism spectrum disorder, and nervous system cancer.InterpretationAs the leading cause of overall disease burden in the world, with increasing global DALY counts, effective prevention, treatment, and rehabilitation strategies for disorders affecting the nervous system are needed

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

Transcriptomic approaches to study the effects of xenobiotics in ruminants

This thesis is concerned with the study and characterization of the xenobiotics-induced transcriptomic signature in some ruminants. Based on the different studies presented in this thesis, the microarray-based transcriptomics approach was able to provide a holistic view on the global gene expression in diverse types of tissues – namely, skeletal muscle, liver, whole blood, primary hepatocytes- and kidney-derived cell lines. The pre-designed commercial bovine microarray enabled the discovery of many biomarkers with which the differentiation between illicitly-treated and untreated veal calves was possible. It also demonstrated the transcriptomic signature dissimilarity between 2 tissues (i.e. skeletal muscle and liver) exposed to the same treatment (i.e. anabolic steroids). Also, the same approach revealed the presence of some transcriptomic landscape convergence between the hepatocytes primary cultures and the Madin-Darby bovine kidney (MDBK) cell line, which in turn spots the light on the MDBK cells as a possible surrogate in vitro tool for some liver-based functional studies. Finally, a custom-designed whole-transcriptome sheep (Ovis aries) microarray revealed the immune-system-induction and the transcriptional-modulation capacity of organic selenium in sheep. Collectively, the transcriptomics approach overcame the shortcoming of focusing on changes in expression of a priori list of selected genes – instead, it looks at the bigger picture within the protein-coding part of the genome. It is important to mention that using an alternative functional analysis tools [i.e. Gene set enrichment analysis (GSEA)] was useful to cross-validate the output of the conventional overrepresentation tools like the Database for Annotation, Visualization and Integrated Discovery (DAVID). The collective body of work represented here shows the adequacy of using microarray, commercial and custom-designed, to depict a holistic picture about the global gene expression profile of a given tissue. Still, there are some challenges in data analysis, interpretation and integration with the output of other alternative omic techniques – those challenges are highlighted and discussed across the different chapters of this thesis.Nella presente tesi sono state caratterizzate le eventuali modulazioni del trascrittoma di alcuni ruminanti conseguentemente all’esposizione a xenobiotici. L'approccio trascrittomico tramite tecnica microarray è stato in grado di fornire una visione olistica sull'espressione genica globale in diversi tipi di tessuti: muscolo scheletrico, fegato, sangue intero, epatociti primari e linee cellulari stabilizzate renali di bovino. Per quanto concerne l’uso di trattamenti illeciti nella filiera del bovino da carne, la tecnica summenzionata ha consentito di individuare un set di biomarcatori potenzialmente in grado di discriminare vitelli trattati illecitamente da quelli di controllo (I) . Inoltre, è stata dimostrata la presenza di pattern di espressione dissimili tra il fegato ed il muscolo scheletrico di animali trattati con steroidi anabolizzanti (II). Questa stessa metodologia omica ha evidenziato una certa similitudine tra il trascrittoma di colture primarie di epatociti e la linea cellulare stabilizzata Madin-Darby bovine kidney (MDBK), che avvalorerebbe la scelta di questa linea cellulare come possibile modello surrogato per studi funzionali sul metabolismo degli xenobiotici nel bovino (III). Infine, è stata designata ‘in-house’ una piattaforma microarray per la caratterizzazione dell’intero trascrittoma dell’ovino (Ovis aries). Grazie a tale piattaforma è stato possibile valutare gli effetti trascrizionali di una dieta addizionata di selenio organico. In particolare, l’addizione di selenio organico è stata in grado di modulare il sistema immunitario dell’ovino (IV). Collettivamente, l'approccio trascrittomico utilizzato ha superato il concetto dell’approccio allo studio di geni candidati selezionati sulla base della letteratura, prediligendo una visuale più ampia dei cambiamenti che avvengono all'interno della parte codificante del genoma. È importante menzionare che l'utilizzo di software di analisi funzionale alternativi quali il Gene Set Enrichment Analysis, GSEA) ha permesso di convalidare l'output ottenuto da software convenzionali come il database per l'annotazione, la visualizzazione e discovery integrato (DAVID). Nel suo insieme, il lavoro qui rappresentato dimostra l'adeguatezza della tecnica microarray, tanto di derivazione commerciale quanto custom-designed, per definire il profilo di espressione genica globale di un individuo, di un particolare tessuto, di un tipo cellulare. Inoltre, nei capitoli di questa tesi vengono discusse alcune sfide riguardanti l’analisi dei dati, la loro interpretazione e loro integrazione con altre tecniche alternative omiche

Transcriptomic characterization of bovine primary cultured hepatocytes; a cross-comparison with a bovine liver and the Madin-Darby bovine kidney cells

Bovine primary cultured hepatocytes (CHs) are widely used in vitro models for liver toxicity testing. However, little is known about their whole-transcriptome profile and its resemblance to the normal liver tissue. In the present study, we profiled \u2013 by microarray - the whole-transcriptome of bovine CHs (n =4) and compared it with the transcriptomic landscape of control liver samples (n =8), as well the Madin-Darby bovine kidney (MDBK) cells (n =4). Compared with liver tissue, the bovine CHs relatively expressed (fold change> 2, P < 0.05) about 2155 and 2073 transcripts at a lower and higher abundance, respectively. Of those expressed at a lower abundance, many were drug biotransformation enzyme-coding genes, such as the cytochrome P450 family (CYPs), sulfotransferases, methyltransferases, and glutathione S-transferases. Also, several drug transporters and solute carriers were expressed at a lower abundance in bovine CHs. \u2018Drug metabolism\u2019, \u2018PPAR signaling\u2019, and \u2018metabolism of xenobiotics by CYPs\u2019 were among the most negatively-enriched pathways in bovine CHs compared with liver. A qPCR cross-validation using 8 selected genes evidenced a high correlation (r =0.95, P=0.001) with the corresponding microarray results. Although from a kidney origin, and albeit to a lower extent compared to bovine CHs, the MDBK cells showed a basal expression of many CYP-coding genes. Our study provides a whole-transcriptome-based evidence for the bovine CHs and hepatic tissue resemblance. Overall, the bovine CHs' transcriptomic profile might render it unreliable as an in vitro model to study drug metabolism

Transcriptomic characterization of Marby Darby Bovine Kidney (MDBK) cell line and its comparison with cattle primary hepatocytes and liver tissue.

INTRODUCTION Cattle hepatocyte primary cultures (CHs) have been used as a useful in vitro model for drug metabolism studies. However, they suffer some limitations, such as a difficult adhesion to the substrate following isolation, a limited viability and a rapid loss of differentiated characteristics if compared to ex vivo liver samples (LSs). Therefore, a reliable hepatic in vitro model is actually needed for cattle. As established bovine hepatic cell lines are not available, we characterized the Marby Darby Bovine Kidney (MDBK) cell line transcriptome by using a cDNA microarray approach, and compared it with CHs and ex vivo cattle LSs. MATERIALS AND METHODS LSs coming from preceding studies, CHs isolated by a two-step collagenase isolation method (Giantin et al., 2012) and MDBK cells collected at different passages were used. Overall, the transcriptome of sixteen samples (8 LSs, 4 CHs and 4 MDBK cell splittings) was characterized by using a cDNA microarray technology. The normalization, statistical analyses, data visualization (Hierarchical clustering and Principal Component Analysis) and functional analyses (gene ontology terms, pathway analysis) of microarray data were performed using the GeneSpring software. RESULTS AND CONCLUSIONS When compared with LSs, CHs showed a significant (P 2.0) of many cell-mediated, immunity-based and metabolic signaling pathways. About drug metabolism, many cytochromes P450 (CYPs), drug transporters (DTs), glutathione transferases and nuclear receptors (NRs) were down-regulated. Likewise, the MDBK cell line showed a significant (P < 0.05) modulation (FC>2.0) of 5307 genes (of which 2706 were down-regulated). Biotransformation and CYP oxidation were among the altered pathways. This study confirms, to a wider scenario (the whole transcriptome), the known decrease of gene expression occurring in CHs, including CYPs, DTs and NRs. MDBK cells, albeit to a lower extent compared to CHs, constitutively express most of drug metabolizing enzymes and DTs. Prospective studies are needed to better characterize the cell line potential as a useful in vitro model for regulation, induction and functional studies. ACKNOWLEDGEMENTS A project supported by the University of Padua (CPDA109434)