Detection of differentially expressed genes in broiler pectoralis major muscle affected by White Striping – Wooden Breast myopathies

Abstract White Striping and Wooden Breast (WS/WB) are abnormalities increasingly occurring in the fillets of high breast yield and growth rate chicken hybrids. These defects lead to consistent economic losses for poultry meat industry, as affected broiler fillets present an impaired visual appearance that negatively affects consumers' acceptability. Previous studies have highlighted in affected fillets a severely damaged muscle, showing profound inflammation, fibrosis, and lipidosis. The present study investigated the differentially expressed genes and pathways linked to the compositional changes observed in WS/WB breast muscles, in order to outline a more complete framework of the gene networks related to the occurrence of this complex pathological picture. The biochemical composition was performed on 20 pectoralis major samples obtained from high breast yield and growth rate broilers (10 affected vs. 10 normal) and 12 out of the 20 samples were used for the microarray gene expression profiling (6 affected vs. 6 normal). The obtained results indicate strong changes in muscle mineral composition, coupled to an increased deposition of fat. In addition, 204 differentially expressed genes (DEG) were found: 102 up-regulated and 102 down-regulated in affected breasts. The gene expression pathways found more altered in WS/WB muscles are those related to muscle development, polysaccharide metabolic processes, proteoglycans synthesis, inflammation, and calcium signaling pathway. On the whole, the findings suggest that a multifactorial and complex etiology is associated with the occurrence of WS/WB muscle abnormalities, contributing to further defining the transcription patterns associated with these myopathies

Time course evaluation of collagen type IV in Pectoralis major muscles of broiler chickens selected for different growth-rates

Collagen type IV (COL4) is one of the major components of animals’ and humans’ basement membranes of several tissues, such as skeletal muscles and vascular endothelia. Alterations in COL4 assembly and secretion are associated to muscular disorders in humans and animals among which growth-related abnormalities such as white striping and wooden breast affecting Pectoralis major muscles (PMs) in modern fast-growing (FG) chickens. Considering the high prevalence of these myopathies in FG broilers and that a worsening is observed as the bird slaughter age is increased, the present study was intended to evaluate the distribution and the expression level of COL4 protein and its coding genes in PMs of FG broilers at different stages of muscle development (i.e., 7, 14, 21, 28, 35, and 42 d of age). Medium-growing (MG) chickens have been considered as the control group in consideration of the lower selection pressure on breast muscle growth rate and hypertrophy. Briefly, 5 PM/sampling time/genotype were selected for western blot, immunohistochemistry (IHC), and gene expression analyses. The normalized expression levels of COL4 coding genes showed an overexpression of COL4A2 in FG than MG at d 28, as well as a significant decrease in its expression over their rearing period. Overall, results obtained through the gene expression analysis suggested that selection for the hypertrophic growth of FG broilers may have led to an altered regulation of fibroblast proliferation and COL4 synthesis. Moreover, western blot and IHC analyses suggested an altered secretion and/or degradation of COL4 protein in FG broilers, as evidenced by the fluctuating trend of 2 bands observed in FG over time. In view of the above, the present research supports the evidence about a potential aberrant synthesis and/or degradation of COL4 and corroborates the hypothesis regarding a likely involvement of COL4 in the series of events underlying the growth-related abnormalities in modern FG broilers

Understanding Marine Biodegradation of Bio-Based Oligoesters and Plasticizers

The study reports the enzymatic synthesis of bio-based oligoesters and chemo-enzymatic processes for obtaining epoxidized bioplasticizers and biolubricants starting from cardoon seed oil. All of the molecules had MW below 1000 g mol-1 and were analyzed in terms of marine biodegradation. The data shed light on the effects of the chemical structure, chemical bond lability, thermal behavior, and water solubility on biodegradation. Moreover, the analysis of the biodegradation of the building blocks that constituted the different bio-based products allowed us to distinguish between different chemical and physicochemical factors. These hints are of major importance for the rational eco-design of new benign bio-based products. Overall, the high lability of ester bonds was confirmed, along with the negligible effect of the presence of epoxy rings on triglyceride structures. The biodegradation data clearly indicated that the monomers/building blocks undergo a much slower process of abiotic or biotic transformations, potentially leading to accumulation. Therefore, the simple analysis of the erosion, hydrolysis, or visual/chemical disappearance of the chemical products or plastic is not sufficient, but ecotoxicity studies on the effects of such small molecules are of major importance. The use of natural feedstocks, such as vegetable seed oils and their derivatives, allows the minimization of these risks, because microorganisms have evolved enzymes and metabolic pathways for processing such natural molecules

The evolution of vimentin and desmin in Pectoralis major muscles of broiler chickens supports their essential role in muscle regeneration

Vimentin (VIM) and desmin (DES) are muscle-specific proteins having crucial roles in maintaining the lateral organization and alignment of the sarcomeric structure during myofibrils’ regeneration. The present experiment was designed to ascertain the evolution of VIM and DES in Pectoralis major muscles (PM) of fast-growing (FG) and medium-growing (MG) meat-type chickens both at the protein and gene levels. MG broilers were considered as a control group whereas the evolution of VIM and DES over the growth period was evaluated in FG by collecting samples at different developmental stages (7, 14, 21, 28, 35, and 42 days). After performing a preliminary classification of the samples based on their histological features, 5 PM/sampling time/genotype were selected for western blot, immunohistochemistry (IHC), and gene expression analyses. Overall, the findings obtained at the protein level mirrored those related to their encoding genes, although a potential time lag required to observe the consequences of gene expression was evident. The two- and 3-fold higher level of the VIM-based heterodimer observed in FG at d 21 and d 28 in comparison with MG of the same age might be ascribed to the beginning and progressive development of the regenerative processes. This hypothesis is supported by IHC highlighting the presence of fibers to coexpressing VIM and DES. In addition, gene expression analyses suggested that, unlike VIM common sequence, VIM long isoform may not be directly implicated in muscle regeneration. As for DES content, the fluctuating trends observed for both the native protein and its heterodimer in FG might be ascribed to its importance for maintaining the structural organization of the regenerating fibers. Furthermore, the higher expression level of the DES gene in FG in comparison with MG further supported its potential application as a marker of muscle fibers’ regeneration. In conclusion, the findings of the present research seem to support the existence of a relationship between the occurrence of muscle regeneration and the growth rate of meat-type chickens and corroborate the potential use of VIM and DES as molecular markers of these cellular processes

Differential expression patterns of genes associated with metabolisms, muscle growth and repair in Pectoralis major muscles of fast- and medium-growing chickens

The aim of this study was to investigate the expression of genes related to muscle growth, hypoxia and oxidative stress responses, a multi-substrate serine/threonine-protein kinase (AMPK) and AMPK-related kinases, carbohydrate metabolism, satellite cells activities and fibro- adipogenic progenitors (FAPs) in fast-growing (FG) (n = 30) and medium-growing (MG) chickens (n = 30). Pectoralis major muscles were collected at 7d, 14d, 21d, 28d, 35d and 42d of age. According to their macroscopic features, the samples from FG up to 21d of age were classified as unaffected, while all samples collected at an older age exhibited macroscopic features ascribable to white striping and/or wooden breast abnormalities. In contrast, MG samples did not show any feature associated to muscle disorders. The absolute transcript abundance of 33 target genes was examined by droplet digital polymerase chain reaction. The results showed differential gene expression profiles between FG and MG chickens at different ages. While most genes remained unchanged in MG chickens, the expression patterns of several genes in FG were significantly affected by age. Genes encoding alpha 1, alpha 2, beta 2 and gamma 3 isoforms of AMPK, as well as AMPK-related kinases, were identified as differentially expressed between the two strains. The results support the hypothesis of oxidative stress-induced muscle damage with metabolic alterations in FG chickens. An increased expression of ANXA2, DES, LITAF, MMP14, MYF5 and TGFB1 was observed in FG strain. The results suggest the occurrence of dysregulation of FAP proliferation and differentiation occurring during muscle repair. FAPs could play an important role in defining the proliferation of connective tissue (fibrosis) and deposition of intermuscular adipose tissue which represents distinctive traits of muscle abnormalities. Overall, these findings demonstrate that dysregulated molecular processes associated with myopathic lesions in chickens are strongly influenced by growth rate, and, to some extent, by age

Enzymatic Processes of the Preparation of Esters of Poorly Water-Soluble Carboxylic Acids

La biodisponibilità di un principio attivo di interesse industriale è il parametro che più influenza la sua traduzione in una molecola bioattiva commerciale. La biodisponibilità è largamente influenzata dalla solubilità in acqua del principio attivo. La maggior parte delle new chemical entities (NCE) sono scarsamente solubili in acqua e quindi scarsamente biodisponibili. Per superare queste limitazioni, è possibile sviluppare protocolli per produrre pro-farmaci che presentino maggiore solubilità in acqua e, quindi, biodisponibilità. Le modificazioni dei principi attivi possono avvenire chimicamente o, in alternativa, enzimaticamente. Questo lavoro propone lo sviluppo di protocolli di esterificazione enzimatica di principi attivi di interesse agroalimentare e farmaceutico. Tra i principi attivi studiati, l'acido sorbico è il conservante più utilizzato nell'industria alimentare. L'inibizione antimicrobica dell'acido sorbico potrebbe essere influenzata dalla sua natura lipofila, che ne riduce l'uso nelle formulazioni alimentari. Le reazioni tra acido sorbico e glicerolo, catalizzate dalle lipasi, sono state studiate al fine di sviluppare un nuovo derivato dell'acido sorbico con un promettente profilo idrofilico. La reazione di esterificazione tra acido sorbico e glicerolo in un sistema privo di solventi è stata eseguita con una Candida antarctica lipasi B immobilizzata (CALB). Tra i principi attivi farmaceutici studiati, l'ibuprofene è un FANS ampiamente utilizzato, poco biodisponibile in quanto scarsamente solubile in acqua. In questa tesi, abbiamo proposto un processo enzimatico ottimizzato per la produzione del profarmaco sorbitol-estere di ibuprofene potenziato in termini di solubilità. L’esterificazione diretta è stata ottimizzata per: scelta del mezzo bifasico, concentrazione di enzima, contenuto di acqua, temperatura, velocità di agitazione, concentrazioni di substrati e tempo di reazione. 1H, 13C-NMR e MS hanno confermato l'esterificazione catalizzata da PPL dell'ibuprofene con sorbitolo. Per quanto a nostra conoscenza, questa è la prima volta che viene proposta l'esterificazione enzimatica dell'ibuprofene con sorbitolo (così come estere di ibuprofene con eritritolo e estere di ibuprofene con xilitolo). Questo processo da il via alla sintesi, con l'approccio biocatalitico, di numerose molecole bioattive con maggiore biodisponibilità di interesse farmaceutico-industriale e agroalimentare. Inoltre, abbiamo sviluppato un protocollo di esterificazione enzimatica emancipato dalla presenza di acqua. Questo protocollo monofasico, per il quale è stato depositato un brevetto, vede l'utilizzo di un unico solvente organico per la solubilizzazione di tutti i substrati in reazione, evitando molte delle limitazioni imposte dai canonici sistemi bifasici O/W. Infine, i profarmaci sintetizzati enzimaticamente sono stati testati per l'attività. Il prodotto glicerolo sorbato è stato testato contro il batterio Streptomices griseus e il lievito Saccharomyces cerevisiae. I risultati indicano che l'esterificazione dell'acido sorbico con glicerolo migliora le sue proprietà antimicrobiche nei confronti di Saccharomyces cerevisie. I risultati riportati dimostrano che l'esterificazione può essere utilizzata come strategia per migliorare l'attività antimicrobica dell'acido sorbico. I derivati dell'ibuprofene sono stati testati in vitro tramite la linea cellulare IB3-1 da pazienti fibrocistici. È stato dimostrato che i derivati mantengono l'attività antinfiammatoria dell'ibuprofene e, in alcuni casi, sembrano migliorarla.The bioavailability of an active ingredient of industrial interest is the parameter that most influences its translation to a marketable bioactive molecule. Bioavailability is largely influenced by the water solubility of the active ingredient. Most of the new chemical entities (NCE) are poorly soluble in water and therefore poorly bioavailable. To overcome these limitations, protocols can be developed to produce pro-drugs with increased solubility in water and, so, bioavailability. The modifications of the active ingredients can take place chemically or, alternatively, enzymatically. This work proposes the development of enzymatic esterification protocols of active ingredients of agri-food and pharmaceutical interest. Among the active ingredients studied, sorbic acid is the most commonly used preservative in the food industry. The antimicrobial inhibition of sorbic acid could be influenced by its lipophilic nature, which reduces its use in hydrophilic food formulations. Reactions between sorbic acid and glycerol catalyzed by lipases were studied in order to develop a novel sorbic acid derivate with a promising hydrophilic profile. The esterification reaction between sorbic acid and glycerol in a solvent-free system was performed with an immobilized lipase B from Candida antarctica (CALB). Among the active pharmaceutical ingredients studied, ibuprofen is the widely used NSAID limited in bioavailability by its poor water solubility. In this thesis, we proposed an effective optimized enzymatic process for the production of the enhanced-water soluble ibuprofen sorbitol ester prodrug. The direct enzymatic route has been optimized for: biphasic media dynamics, enzyme concentration, water content, temperature, stirring speed, substrates concentrations, and reaction time. 1H, 13C-NMR, and MS confirmed the PPL-catalyzed esterification of ibuprofen with sorbitol. To the best of our knowledge, this is the first time that enzymatic esterification of ibuprofen with sorbitol (as well as ibuprofen erythritol ester, and xylitol ester), has been proposed. This process initiates the synthesis with the biocatalytic approach of numerous bioactive molecules with increased bioavailability of pharmaceutical-industrial and agri-food interest. Moreover, we have developed an enzymatic esterification protocol emancipated from the presence of water. This monophasic protocol, for which a patent has been deposited, sees the use of a single organic solvent for the solubilization of all substrates in reaction, avoiding many of the limitations imposed by the canonical O/W biphasic systems. Finally, the enzymatically synthesized prodrugs were tested for activity. The glycerol sorbate product has been tested against Streptomices griseus bacterium and Saccharomyces cerevisiae yeast. Results indicate that the esterification of sorbic acid with glycerol does improve its antimicrobial properties against Saccharomyces cerevisie. The reported results demonstrate that esterification can be used as a strategy to improve the antimicrobial activity of sorbic acid. Ibuprofen derivatives were tested in vitro via the IB3-1 cell line from fibrocystic patients. The derivatives have been shown to maintain the anti-inflammatory activity of ibuprofen and, in some cases, appear to improve it

Deign of Positive Displacement Gear Machine-based Electro-hydraulic Units.pdf

In recent years increasingly stringent regulations regarding the pollution emissions and greenhouse gasses (GHG) of off-highway vehicles have emerged. However, recent studies underscores that off-highway vehicles have an average efficiency of 30%. In response, researchers are exploring the possibility of electrifying these vehicles with electric machines (EMs) potentially undertaking one, multiple, or all the vehicular functions previously reliant on internal combustion engines (ICEs).Contemporary off-highway vehicle technology revolves around hydraulic systems tailored for diesel engines, tuned to specific torque characteristics and operating at a single speed. While replacing the prime mover with electric machines, the proper hydraulic supply capable of matching the same torque speed characteristics must be found. Furthermore, it must be determined whether an integration capable of reducing the mass, cost, and volume can be implemented, and if energy recuperation is possible. In essence, achieving the desired transformation in off-highway vehicle technology necessitates a comprehensive reevaluation of both hydraulic systems and power sources, with electrification emerging as a promising strategy for harmonizing efficiency, emission standards, and performance expectations. This work proposes guidelines to systematically design EMs and positive displacement hydraulic gear machines (HMs), along with their integration in an electro-hydraulic unit (EHU). To do so, three different variants of EHU are produced. The first variant features an external gear machine (EGM) integrated in a permanent magnet synchronous electric machine (PMSEM). The second and third variants integrate an internal gear machine (IGM) and a PMSEM, wherein the final variant introduces features endowing its operation at high rotational velocities.The EM and HM constituting all variants of EHU are designed using a genetic algorithm-based optimization framework. This optimization framework encapsulates dedicated models for the EM and the HM that allow the calculation of the EHU performance. The first optimization objectives are the minimization of power consumption over the duty cycle of the selected reference machine, the minimization of the pressure and flow ripple, and maximization of the power density of the EHU. The optimization of the second and third variants instead only aims to maximize the total efficiency and power density of the EHU. After having determined the parameters of the EHU through the optimization procedure the designs are refined with thorough simulations focusing on the fluid-dynamic features and the design of the axial balance system of the HMs. The three variants present an increasing level of HM and EM integration and component reduction. While in the first variant HM and EM have a dedicated housing, and the HM is only positioned in the inert region of the EM, in the latest variants the HM and the EM also share the same casing. The first variant of EHU is air cooled by a radial fan system attached to the EM rotor and openings machined in the casing. The second variant takes advantage of the extreme integration and the differential pressure generated across the HM to liquid cooling the EM. The third variant necessitates the use of an external system to cool the EM. To prove the effectiveness of the design process the first two EHU variants are prototyped and tested. The first EHU variant is tested both in a standalone configuration and on the reference machine showing total efficiency values up to 69%, proving its functionality and proving the capability of recuperating energy. The tests conducted on the second variant EHU show a volumetric efficiency that ranges between 81% and above 96% for a pinion rotation velocity of 6000 rpm proving the value of the presented design process. Despite the good quality of the volumetric efficiency values, this EHU variant present morphological limitations that negatively impact its mechanical efficiency. Finally, the third EHU concept is presented not only to remedy the morphological limitations of the second variant but also to address the challenges raised by high rotational velocity operation. </p

Fermentation as a Strategy for Bio-Transforming Waste into Resources: Lactic Acid Production from Agri-Food Residues

Lactic acid (LA) obtained by fermentation of carbohydrates is well-known and widely used in the food sector. This process is as an alternative to the chemical synthesis and ensures several advantages especially in terms of environmental sustainability. In particularly, the opportunity to use agro-food residues as fermentable raw materials could improve the overall process sustainability, without considering the indisputable advantages in terms of waste reduction and residual biomass valorization, in a bio- and circular economy perspective. This research deals with the study and development of the fermentation processes of various waste biomasses from the agro-food industries, including milk whey (MW), ricotta cheese whey (RCW), pear processing residues (PPR), potato pomace (PP), tomato pomace (PT), in order to obtain an experimental protocol applicable to the production of LA. Lactobacillus casei DSM 20011 (ATCC 393), a homofermentative L(+)-LA producing bacterium has been used, starting from small-scale tests to verify of the microorganism to grow in complex medium with different carbon sources and the possible presence of potentially toxic substances for microbial growth. Yields from 27.0 ± 0.3% to 46.0 ± 0.7% have been obtained. Then, a scaling-up was performed in a 1 L batch fermenter, using a mixed medium of RCW and PPR in different ratio. The best LA yield was 78.3% with a volumetric productivity of 1.12 g/L·h in less than 60 h

Design of Electro-Hydraulic Drive for Off-Road Vehicles

Electrification is an increasing trend in developing the next generation of large-scale off-road vehicles. Benefits of electrification are high operating efficiency, low emissions, the possibility of using energy coming from renewable sources, and improved controllability. Despite the growth of these technologies there is no clear direction for how the system should be electrified. This research focuses on defining a standardized methodology to design different electro-hydraulic units that can function as a prime mover for a wide variety of applications. The electro-hydraulic units are designed using a genetic algorithm-based optimization that couples one model dedicated to the hydraulic machine and one model dedicated to the electric machine and determines the optimal parameters of the electro-hydraulic unit given a specific application. After having designed an external gear-type, internal gear-type, and an axial piston-type hydraulic machine, several prototypes are produced, and tested on a reference machine

CRISPR activation on coagulation F7 or F8 promoters potentiate trascriptional activity in the normal and mutated gene context

Background: Engineered transcription factors (eTF) have been successfully exploited to modulate gene expression and represent potential therapeutic tools for human disorders. In this perspective, the emerging CRISPR activation (CRISPRa) technology gives great advantages compared to the first eTF, mostly based on Transcription-Activator-like Effectors (TALE). Coagulation factor disorders, in which even modest protein level increase have a therapeutic impact, represent ideal models to test the CRISPRa. Aims: To tailor the CRISPRa system on the promoters of coagulation F7/F8 genes and enhance transcription, and thus expression, in the normal and mutated conditions. Methods: Creation of reporter gene (luciferase) constructs for F7 and F8 promoter. Transient transfection in hepatoma cells and evaluation of the luciferase activity. Evaluation of the FVIII expression by functional assays (FXa generation). Results: Using reporter gene assays we identified a sgRNA able to trigger the activity of F7 promoter up to ~35-fold, either wild-type, or defective due to the disease-causing c.-61T&gt;G mutation. The effect was higher than that of an engineered TALE-based effector targeting the same promoter region (~15-fold). The transcription increase was confirmed on the endogenous F7 gene, the dCas9-VPR/sgRNAF7.5 combination was more efficient (~6.5-fold) in promoting factor VII (FVII) protein secretion/activity than TALE-TF4 (~3.8-fold). The approach was translated on F8 promoter, whose reduced expression translates into Haemophilia A. Reporter gene assays identified sgRNAs that appreciably increased F8 promoter activity (sgRNAF8.1, ~8-fold; sgRNAF8.2, ~19-fold) with a synergistic effect (~38-fold) when combined. Conclusions: Over this pioneer study we demonstrated that the CRISPR system can be addressed to increase the expression, or rescue disease-causing mutations, of different promoters, with potential intriguing implications for coagulopathies