Molecular Aspects of the Functioning of Pathogenic Bacteria Biofilm Based on Quorum Sensing (QS) Signal-Response System and Innovative Non-Antibiotic Strategies for Their Elimination.

One of the key mechanisms enabling bacterial cells to create biofilms and regulate crucial life functions in a global and highly synchronized way is a bacterial communication system called quorum sensing (QS). QS is a bacterial cell-to-cell communication process that depends on the bacterial population density and is mediated by small signalling molecules called autoinducers (AIs). In bacteria, QS controls the biofilm formation through the global regulation of gene expression involved in the extracellular polymeric matrix (EPS) synthesis, virulence factor production, stress tolerance and metabolic adaptation. Forming biofilm is one of the crucial mechanisms of bacterial antimicrobial resistance (AMR). A common feature of human pathogens is the ability to form biofilm, which poses a serious medical issue due to their high susceptibility to traditional antibiotics. Because QS is associated with virulence and biofilm formation, there is a belief that inhibition of QS activity called quorum quenching (QQ) may provide alternative therapeutic methods for treating microbial infections. This review summarises recent progress in biofilm research, focusing on the mechanisms by which biofilms, especially those formed by pathogenic bacteria, become resistant to antibiotic treatment. Subsequently, a potential alternative approach to QS inhibition highlighting innovative non-antibiotic strategies to control AMR and biofilm formation of pathogenic bacteria has been discussed.

Newly Isolated Virulent Salmophages for Biocontrol of Multidrug-Resistant Salmonella in Ready-to-Eat Plant-Based Food.

Due to irrational antibiotic stewardship, an increase in the incidence of multidrug resistance of bacteria has been observed recently. Therefore, the search for new therapeutic methods for pathogen infection treatment seems to be necessary. One of the possibilities is the utilization of bacteriophages (phages)-the natural enemies of bacteria. Thus, this study is aimed at the genomic and functional characterization of two newly isolated phages targeting MDR Salmonella enterica strains and their efficacy in salmonellosis biocontrol in raw carrot-apple juice. The Salmonella phage vB_Sen-IAFB3829 (Salmonella phage strain KKP 3829) and Salmonella phage vB_Sen-IAFB3830 (Salmonella phage strain KKP 3830) were isolated against S. I (6,8:l,-:1,7) strain KKP 1762 and S. Typhimurium strain KKP 3080 host strains, respectively. Based on the transmission electron microscopy (TEM) and whole-genome sequencing (WGS) analyses, the viruses were identified as members of tailed bacteriophages from the Caudoviricetes class. Genome sequencing revealed that these phages have linear double-stranded DNA and sizes of 58,992 bp (vB_Sen-IAFB3829) and 50,514 bp (vB_Sen-IAFB3830). Phages retained their activity in a wide range of temperatures (from -20 °C to 60 °C) and active acidity values (pH from 3 to 11). The exposure of phages to UV radiation significantly decreased their activity in proportion to the exposure time. The application of phages to the food matrices significantly reduced the level of Salmonella contamination compared to the control. Genome analysis showed that both phages do not encode virulence or toxin genes and can be classified as virulent bacteriophages. Virulent characteristics and no possible pathogen factors make examined phages feasible to be potential candidates for food biocontrol.

Effect of in vitro cultivation on human gut microbiota composition using 16S rDNA amplicon sequencing and metabolomics approach.

Gut microbiota (GM) plays many key functions and helps maintain the host's health. Consequently, the development of GM cultivation under in vitro stimulating physiological conditions has gained extreme interest in different fields. In this study, we evaluated the impact of four culture media: Gut Microbiota Medium (GMM), Schaedler Broth (SM), Fermentation Medium (FM), and Carbohydrate Free Basal Medium (CFBM) on preserving the biodiversity and metabolic activity of human GM in batch in vitro cultures using PMA treatment coupled with 16S rDNA sequencing (PMA-seq) and LC-HR-MS/MS untargeted metabolomics supplemented with GC-MS SCFA profiling. Before the experiments, we determined the possibility of using the pooled faecal samples (MIX) from healthy donors (n = 15) as inoculum to reduce the number of variables and ensure the reproducibility of in vitro cultivation tests. Results showed the suitability of pooling faecal samples for in vitro cultivation study. Non-cultured MIX inoculum was characterized by higher α-diversity (Shannon effective count, and Effective microbial richness) compared to inocula from individual donors. After 24 h of cultivation, a significant effect of culture media composition on GM taxonomic and metabolomic profiles was observed. The SM and GMM had the highest α-diversity (Shannon effective count). The highest number of core ASVs (125) shared with non-cultured MIX inoculum and total SCFAs production was observed in the SM. These results might contribute to the development of standardized protocols for human GM in vitro cultivation by preventing methodological bias in the data.

Effectiveness of a Phage Cocktail as a Potential Biocontrol Agent against Saprophytic Bacteria in Ready-To-Eat Plant-Based Food.

This study aimed to evaluate the effectiveness of the phage cocktail to improve the microbiological quality of five different mixed-leaf salads: rucola, mixed-leaf salad with carrot, mixed-leaf salad with beetroot, washed and unwashed spinach, during storage in refrigerated conditions. Enterobacterales rods constituted a significant group of bacteria in the tested products. Selected bacteria were tested for antibiotic resistance profiles and then used to search for specific bacteriophages. Forty-three phages targeting bacteria dominant in mixed-leaf salads were isolated from sewage. Their titer was determined, and lytic activity was assessed using the Bioscreen C Pro automated growth analyzer. Two methods of phage cocktail application including spraying, and an absorption pad were effective for rucola, mixed leaf salad with carrot, and mixed leaf salad with beetroot. The maximum reduction level after 48 h of incubation reached 99.9% compared to the control sample. In washed and unwashed spinach, attempts to reduce the number of microorganisms did not bring the desired effect. The decrease in bacteria count in the lettuce mixes depended on the composition of the autochthonous saprophytic bacteria species. Both phage cocktail application methods effectively improved the microbiological quality of minimally processed products. Whole-spectral phage cocktail application may constitute an alternative food microbiological quality improvement method without affecting food properties.

Application of In Vitro Models for Studying the Mechanisms Underlying the Obesogenic Action of Endocrine-Disrupting Chemicals (EDCs) as Food Contaminants-A Review.

Obesogenic endocrine-disrupting chemicals (EDCs) belong to the group of environmental contaminants, which can adversely affect human health. A growing body of evidence supports that chronic exposure to EDCs can contribute to a rapid increase in obesity among adults and children, especially in wealthy industrialized countries with a high production of widely used industrial chemicals such as plasticizers (bisphenols and phthalates), parabens, flame retardants, and pesticides. The main source of human exposure to obesogenic EDCs is through diet, particularly with the consumption of contaminated food such as meat, fish, fruit, vegetables, milk, and dairy products. EDCs can promote obesity by stimulating adipo- and lipogenesis of target cells such as adipocytes and hepatocytes, disrupting glucose metabolism and insulin secretion, and impacting hormonal appetite/satiety regulation. In vitro models still play an essential role in investigating potential environmental obesogens. The review aimed to provide information on currently available two-dimensional (2D) in vitro animal and human cell models applied for studying the mechanisms of obesogenic action of various industrial chemicals such as food contaminants. The advantages and limitations of in vitro models representing the crucial endocrine tissue (adipose tissue) and organs (liver and pancreas) involved in the etiology of obesity and metabolic diseases, which are applied to evaluate the effects of obesogenic EDCs and their disruption activity, were thoroughly and critically discussed.

Bacterial Pathogens in the Food Industry: Antibiotic Resistance and Virulence Factors of Salmonella enterica Strains Isolated from Food Chain Links.

Salmonella is one of the most important foodborne pathogens. Fifty-three strains of Salmonella deposited in the Culture Collection of Industrial Microorganisms-Microbiological Resources Center (IAFB) were identified using molecular and proteomic analyses. Moreover, the genetic similarity of the tested strains was determined using the PFGE method. Main virulence genes were identified, and phenotypical antibiotic susceptibility profiles and prevalence of resistance genes were analyzed. Subsequently, the occurrence of the main mechanisms of ß-lactam resistance was determined. Virulence genes, invA, fimA, and stn were identified in all tested strains. Phenotypic tests, including 28 antibiotics, showed that 50.9% of the strains were MDR. The tet genes associated with tetracyclines resistance were the most frequently identified genes. Concerning the genes associated with ESBL-producing Salmonella, no resistance to the TEM and CTX-M type was identified, and only two strains (KKP 1597 and KKP 1610) showed resistance to SHV. No strains exhibited AmpC-type resistance but for six Salmonella strains, the efflux-related resistance of PSE-1 was presented. The high number of resistant strains in combination with multiple ARGs in Salmonella indicates the possible overuse of antibiotics. Our results showed that it is necessary to monitor antimicrobial resistance profiles in all food chain links constantly and to implement a policy of proper antibiotic stewardship to contain or at least significantly limit the further acquisition of antibiotic resistance among Salmonella strains.

Molecular Mechanism of Equine Endometrosis: The NF-κB-Dependent Pathway Underlies the Ovarian Steroid Receptors' Dysfunction.

Endometrosis is a frequently occurring disease decreasing mares' fertility. Thus, it is an important disease of the endometrium associated with epithelial and stromal cell alterations, endometrium gland degeneration and periglandular fibrosis. Multiple degenerative changes are found in uterine mucosa, the endometrium. However, their pathogenesis is not well known. It is thought that nuclear factor-κB (NF-κB), a cell metabolism regulator, and its activation pathways take part in it. The transcription of the profibrotic pathway genes of the NF-κB in fibrotic endometria differed between the follicular (FLP) and mid-luteal (MLP) phases of the estrous cycle, as well as with fibrosis progression. This study aimed to investigate the transcription of genes of estrogen (ESR1, ESR2) and progesterone receptors (PGR) in equine endometria to find relationships between the endocrine environment, NF-κB-pathway, and fibrosis. Endometrial samples (n = 100), collected in FLP or MLP, were classified histologically, and examined using quantitative PCR. The phase of the cycle was determined through the evaluation of ovarian structures and hormone levels (estradiol, progesterone) in serum. The transcription of ESR1, ESR2, and PGR decreased with the severity of endometrial fibrosis and degeneration of the endometrium. Moreover, differences in the transcription of ESR1, ESR2, and PGR were noted between FLP and MLP in the specific categories and histopathological type of equine endometrosis. In FLP and MLP, specific moderate and strong correlations between ESR1, ESR2, PGR and genes of the NF-κB pathway were evidenced. The transcription of endometrial steroid receptors can be subjected to dysregulation with the degree of equine endometrosis, especially in both destructive types of endometrosis, and mediated by the canonical NF-κB pathway depending on the estrous cycle phase.

The NF-κB-signalling pathway in mare's endometrium infiltrated with the inflammatory cells.

Endometritis is an important issue decreasing mares' fertility. In the case of endometritis, both inflammatory cells infiltration and proinflammatory molecules production are regulated by various cellular and gene regulatory mechanisms, including the nuclear factor-κB (NF-κB)-dependent pathway. NF-κB-signalling pathway has been recently studied in the equine endometrium in the context of endometrosis. Thus, this study aimed to determine gene transcription of NF-κB subunits (RelA; NF-κB1; NF-κB2), proinflammatory molecules (MCP-1; IL-6) and hyaluronan synthases (HAS 1; HAS 2; HAS 3) in endometritis and compare them with the intensity and type of inflammatory cell infiltration. Endometrial samples, collected post-mortem from cyclic mares in oestrus or dioestrus, were classified histologically and examined using quantitative PCR. Transcription NF-κB subunits genes did not differ with either inflammatory intensity or type of inflammatory cell infiltration. Transcription of MCP-1 and IL-6 genes increased with the severity of inflammation, with the involvement of HAS 3 and HAS 2 genes, as opposed to HAS 1 genes. These proinflammatory molecules and hyaluronan synthases in the equine inflamed endometrium do not seem to be regulated by the NF-κB pathway. Hence, separate signalling pathways for the development and progression of equine endometritis and endometrosis may be suggested.

Equine Endometrosis Pathological Features: Are They Dependent on NF-κB Signaling Pathway?

Endometrosis is an important mares' disease which considerably decreases their fertility. As classic endometrial classification methods might be insufficient for tissue pathological evaluation, further categorization into active/inactive and destructive/non-destructive types was developed by Hoffmann and others. This study aimed to compare NF-κB pathway genes transcription among histopathological types of endometrosis, following Hoffmann and co-authors' classification. Endometrial samples, collected postmortem from cyclic mares (n = 100) in estrus or diestrus, were classified histologically and used for gene transcription assessment. Gene transcription of NF-κB subunits (RelA, NF-κB1, NF-κB2), pro-inflammatory molecules (MCP-1, IL-6), and hyaluronan synthases (HAS 1, HAS 2, HAS 3) was compared among endometrosis types (active, non-active, destructive, non-destructive). Most individual mRNA samples showed high expression of RelA, NF-κB1, and MCP-1 gene transcripts and the destructive type of endometrosis, simultaneously. The expression of RelA and NF-κB1 genes was higher in active destructive group than in the other groups only in the follicular phase, as well as being higher in the inactive destructive group than in the others, only in the mid-luteal phase. The increase in gene transcription of the NF-κB canonical activation pathway in destructive endometrosis may suggest the highest changes in extracellular matrix deposition. Moreover, the estrous cycle phase might influence fibrosis pathogenesis.

Effect of High Hydrostatic Pressure on Stress-Related dnaK, hrcA, and ctsR Expression Patterns in Selected Lactobacilli Strains.

Lactic acid bacteria (LAB) in the natural environment meet multiple stressors such as pH and temperature variations, increased nutrition and metabolite concentrations, harmful chemicals, acidic/oxidative conditions, osmotic pressure, and starvation. However, LAB strains are not subjected to high hydrostatic pressure (HHP) which currently is the most common non-thermal decontamination technology in the food industry. In this context, the LAB response to HHP is more difficult to identify compared to other stress-induced responses, and dnaK, ctsR, and hrcA can serve as essential regulators in this reaction. In the present study, the expression level of dnaK, ctsR, and hrcA mRNAs in 15 LAB strains after the HHP (300 MPa/5') exposure was evaluated. As a result, the HHP-treatment affected the up-regulation of dnaK, ctsR, and hrcA in L. backii KKP 3565, L. backii KKP 3566, L. rhamnosus KKP 3570, L. brevis KKP 3575 strains, whereas, in L. plantarum KKP 3569, L. rhamnosus KKP 3571, L. brevis KKP 3573 all genes were lower expressed. The relative expression level of the dnaK, ctsR, and hrcA either before or after the pressure treatment for L. brevis DSM 6235, L. rhamnosus KKP 3572, L. brevis KKP 3574, L. brevis KKP 3576, L. rossiae KKP 3577, L. curvatus KKP 3578 strains were undetectable. Significant differences in the expression levels were observed, between the control and the HHP treatment strains for dnaK in L. backii KKP 3565, L. backii KKP 3566, L. plantarum KKP 3569, L. rhamnosus KKP 3570, L. rhamnosus KKP 3571, ctsR in, L. backii KKP 3565, L. rhamnosus KKP 3570, L. rhamnosus KKP 3571, and hrcA in L. plantarum KKP 3569, L. rhamnosus KKP 3571. Overall, the studied genes, dnaK, ctsR, and hrcA can be useful markers to indicate the LAB cellular response to HHP. These molecular parameters can help to optimize the desirable LAB growing conditions in industrial processes and to understand the complexity of the stress-related mechanism.

Characterization and Genome Study of Novel Lytic Bacteriophages against Prevailing Saprophytic Bacterial Microflora of Minimally Processed Plant-Based Food Products.

The food industry is still searching for novel solutions to effectively ensure the microbiological safety of food, especially fresh and minimally processed food products. Nowadays, the use of bacteriophages as potential biological control agents in microbiological food safety and preservation is a promising strategy. The aim of the study was the isolation and comprehensive characterization of novel bacteriophages with lytic activity against saprophytic bacterial microflora of minimally processed plant-based food products, such as mixed leaf salads. From 43 phages isolated from municipal sewage, four phages, namely Enterobacter phage KKP 3263, Citrobacter phage KKP 3664, Enterobacter phage KKP 3262, and Serratia phage KKP 3264 have lytic activity against Enterobacter ludwigii KKP 3083, Citrobacter freundii KKP 3655, Enterobacter cloacae KKP 3082, and Serratia fonticola KKP 3084 bacterial strains, respectively. Transmission electron microscopy (TEM) and whole-genome sequencing (WGS) identified Enterobacter phage KKP 3263 as an Autographiviridae, and Citrobacter phage KKP 3664, Enterobacter phage KKP 3262, and Serratia phage KKP 3264 as members of the Myoviridae family. Genome sequencing revealed that these phages have linear double-stranded DNA (dsDNA) with sizes of 39,418 bp (KKP 3263), 61,608 bp (KKP 3664), 84,075 bp (KKP 3262), and 148,182 bp (KKP 3264). No antibiotic resistance genes, virulence factors, integrase, recombinase, or repressors, which are the main markers of lysogenic viruses, were annotated in phage genomes. Serratia phage KKP 3264 showed the greatest growth inhibition of Serratia fonticola KKP 3084 strain. The use of MOI 1.0 caused an almost 5-fold decrease in the value of the specific growth rate coefficient. The phages retained their lytic activity in a wide range of temperatures (from -20 °C to 50 °C) and active acidity values (pH from 4 to 11). All phages retained at least 70% of lytic activity at 60 °C. At 80 °C, no lytic activity against tested bacterial strains was observed. Serratia phage KKP 3264 was the most resistant to chemical factors, by maintaining high lytic activity across a broader range of pH from 3 to 11. The results indicated that these phages could be a potential biological control agent against saprophytic bacterial microflora of minimally processed plant-based food products.

Time Evolution of Microbial Composition and Metabolic Profile for Biogenic Amines and Free Amino Acids in a Model Cucumber Fermentation System Brined with 0.5% to 5.0% Sodium Chloride.

Salt concentrations in brine and temperature are the major environmental factors that affect activity of microorganisms and, thus may affect formation of biogenic amines (BAs) during the fermentation process. A model system to ferment cucumbers with low salt (0.5%, 1.5% or 5.0% NaCl) at two temperatures (11 or 23 °C) was used to study the ability of indigenous microbiota to produce biogenic amines and metabolize amino acid precursors. Colony counts for presumptive Enterococcus and Enterobacteriaceae increased by 4 and up to 2 log of CFU∙mL-1, respectively, and remained viable for more than 10 days. 16S rRNA sequencing showed that Lactobacillus and Enterobacter were dominant in fermented cucumbers with 0.5% and 1.5% salt concentrations after storage. The initial content of BAs in raw material of 25.44 ± 4.03 mg∙kg-1 fluctuated throughout experiment, but after 6 months there were no significant differences between tested variants. The most abundant BA was putrescine, that reached a maximum concentration of 158.02 ± 25.11 mg∙kg-1. The Biogenic Amines Index (BAI) calculated for all samples was significantly below that needed to induce undesirable effects upon consumption. The highest value was calculated for the 23 °C/5.0% NaCl brine variant after 192 h of fermentation (223.93 ± 54.40). Results presented in this work indicate that possibilities to control spontaneous fermentation by changing salt concentration and temperature to inhibit the formation of BAs are very limited.

Transcriptional Regulation of the Multiple Resistance Mechanisms in Salmonella-A Review.

The widespread use of antibiotics, especially those with a broad spectrum of activity, has resulted in the development of multidrug resistance in many strains of bacteria, including Salmonella. Salmonella is among the most prevalent causes of intoxication due to the consumption of contaminated food and water. Salmonellosis caused by this pathogen is pharmacologically treated using antibiotics such as fluoroquinolones, ceftriaxone, and azithromycin. This foodborne pathogen developed several molecular mechanisms of resistance both on the level of global and local transcription modulators. The increasing rate of antibiotic resistance in Salmonella poses a significant global concern, and an improved understanding of the multidrug resistance mechanisms in Salmonella is essential for choosing the suitable antibiotic for the treatment of infections. In this review, we summarized the current knowledge of molecular mechanisms that control gene expression related to antibiotic resistance of Salmonella strains. We characterized regulators acting as transcription activators and repressors, as well as two-component signal transduction systems. We also discuss the background of the molecular mechanisms of the resistance to metals, regulators of multidrug resistance to antibiotics, global regulators of the LysR family, as well as regulators of histone-like proteins.

Molecular Characterisation of Uterine Endometrial Proteins during Early Stages of Pregnancy in Pigs by MALDI TOF/TOF.

The molecular mechanism underlying embryonic implantation is vital to understand the correct communications between endometrium and developing conceptus during early stages of pregnancy. This study's objective was to determine molecular changes in the uterine endometrial proteome during the preimplantation and peri-implantation between 9 days (9D), 12 days (12D), and 16 days (16D) of pregnant Polish Large White (PLW) gilts. 2DE-MALDI-TOF/TOF and ClueGOTM approaches were employed to analyse the biological networks and molecular changes in porcine endometrial proteome during maternal recognition of pregnancy. A total of sixteen differentially expressed proteins (DEPs) were identified using 2-DE gels and MALDI-TOF/TOF mass spectrometry. Comparison between 9D and 12D of pregnancy identified APOA1, CAPZB, LDHB, CCT5, ANXA4, CFB, TTR upregulated DEPs, and ANXA5, SMS downregulated DEPs. Comparison between 9D and 16D of pregnancy identified HP, APOA1, ACTB, CCT5, ANXA4, CFB upregulated DEPs and ANXA5, SMS, LDHB, ACTR3, HP, ENO3, OAT downregulated DEPs. However, a comparison between 12D and 16D of pregnancy identified HP, ACTB upregulated DEPs, and CRYM, ANXA4, ANXA5, CAPZB, LDHB, ACTR3, CCT5, ENO3, OAT, TTR down-regulated DEPs. Outcomes of this study revealed key proteins and their interactions with metabolic pathways involved in the recognition and establishment of early pregnancy in PLW gilts.

Probiotics as a biological detoxification tool of food chemical contamination: A review.

Nowadays, people are exposed to diverse environmental and chemical pollutants produced by industry and agriculture. Food contaminations such as persistent organic pollutants (POPs), heavy metals, and mycotoxins are a serious concern for global food safety with economic and public health implications especially in the newly industrialized countries (NIC). Mounting evidence indicates that chronic exposure to food contaminants referred to as xenobiotics exert a negative effect on human health such as inflammation, oxidative stress, and intestinal disorders linked with perturbation of the composition and metabolic profile of the gut microflora. Although the physicochemical technologies for food decontamination are utilized in many cases but require adequate conditions which are often not feasible to be met in many industrial sectors. At present, one promising approach to reduce the risk related to the presence of xenobiotics in foodstuffs is a biological detoxification done by probiotic strains and their enzymes. Many studies confirmed that probiotics are an effective, feasible, and inexpensive tool for preventing xenobiotic-induced dysbiosis and alleviating their toxicity. This review aims to summarize the current knowledge of the direct mechanisms by which probiotics can influence the detoxification of xenobiotics. Moreover, probiotic-xenobiotic interactions with the gut microbiota and the host response were also discussed.

Expression of genes involved in the NF-κB-dependent pathway of the fibrosis in the mare endometrium.

Equine endometrosis is a multifactorial chronic degenerative condition, considered to be one of a major causes of equine infertility. The formation of periglandular fibrosis seems to be linked to chronic inflammation of the mare endometrium in a paracrine way and in a response to numerous forms of inflammatory stimuli elicit the net deposition of extracellular matrix (ECM) around the endometrial glands and stroma. We hypothesized some of these stimuli, such as monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and hyaluronan synthases (HASs), may share the nuclear factor-κB (NF-κB) dependent activation pathway. This study aimed to determine whether mRNA expression of MCP-1, IL-6, HASs, and proteins of canonical (RelA/NK-κß1) and noncanonical (NK-κß2) signaling pathways for NF-kB would change in subsequent categories of endometrosis during the estrous cycle. The expression of selected genes was established in mare endometrium (n = 80; Kenney and Doig categories I, IIA, IIB, III), obtained in the follicular phase (FLP) and mid-luteal phase (MLP). The high expression of RelA mRNA was observed in III, whereas of NK-κß1 and NK-κß2 also in IIA, and IIA and IIB, respectively. The expression of MCP-1 mRNA occurred constantly, regardless of the category, whereas IL-6 mRNA was low in IIA, IIB, and III. The expression of HAS 1 was high in IIA and HAS 3 in IIA, IIB, and III. All those changes were observed in FLP, but not MLP. Our results suggest that NF-κB may be involved in progression of the chronic degenerative condition of the mare endometrium, on both canonical and noncanonical pathways. The most important changes in target genes expression were observed only in FLP, which may suggest the hormone-dependent activation of the NF-κB-dependent fibrosis pathway.

Acute-phase proteins, cortisol and haematological parameters in ewes during the periparturient period.

Numerous subclinical diseases in sheep occur in the periparturient period and involve inflammatory processes; therefore, determining markers, such as acute-phase proteins (APPs), can allow an early diagnosis. Therefore, the objective of the present study was to assess changes in the plasma concentration of APPs and cortisol in clinically healthy ewes in the periparturient period for use in future studies. At the same time, haematological parameters were monitored. We showed that plasma APPs and cortisol concentrations were significantly higher in pregnant ewes than before insemination. A gradual increase in the SAA concentration was observed from the 14th day before to the day of parturition, while Hp was reduced from 2 weeks before up to 2 weeks after delivery. A significant increase in the Fb concentration was detected from the 14th day before to the 1st week after delivery. The cortisol concentration did not undergo significant changes in the periparturient period. We found an increase in the SAA and Fb concentrations and decrease in Hp in the periparturient period. The direction of the change in APPs of healthy ewes in the current study may be related to their distinct regulatory mechanisms during pregnancy. The APPs are usually altered during infection, inflammation, neoplasia, stress and trauma; therefore, knowing their reference values could help lead to an early diagnosis of subclinical forms of some diseases and pregnancy complications in ewes. The haematological analysis showed that ewes in late pregnancy and postpartum compared to dry period were under metabolic stress related to pregnancy and lactation.

Comparative Analysis of the Liver Transcriptome among Cattle Breeds Using RNA-seq.

Global gene expression in liver transcriptome varies among cattle breeds. The present investigation was aimed to identify the differentially expressed genes (DEGs), metabolic gene networks and metabolic pathways in bovine liver transcriptome of young bulls. In this study, we comparatively analyzed the bovine liver transcriptome of dairy (Polish Holstein Friesian (HF); n = 6), beef (Hereford; n = 6), and dual purpose (Polish-Red; n = 6) cattle breeds. This study identified 895, 338, and 571 significant (p < 0.01) differentially expressed (DE) gene-transcripts represented as 745, 265, and 498 hepatic DE genes through the Polish-Red versus Hereford, Polish-HF versus Hereford, and Polish-HF versus Polish-Red breeds comparisons, respectively. By combining all breeds comparisons, 75 hepatic DE genes (p < 0.01) were identified as commonly shared among all the three breed comparisons; 70, 160, and 38 hepatic DE genes were commonly shared between the following comparisons: (i) Polish-Red versus Hereford and Polish-HF versus Hereford; (ii) Polish-Red versus Hereford and Polish-HF versus Polish-Red; and (iii) Polish-HF versus Hereford and Polish-HF versus Polish-Red, respectively. A total of 440, 82, and 225 hepatic DE genes were uniquely observed for the Polish-Red versus Hereford, Polish-HF versus Hereford, and Polish-Red versus Polish-HF comparisons, respectively. Gene ontology (GO) analysis identified top-ranked enriched GO terms (p < 0.01) including 17, 16, and 31 functional groups and 151, 61, and 140 gene functions that were DE in all three breed liver transcriptome comparisons. Gene network analysis identified several potential metabolic pathways involved in glutamine family amino-acid, triglyceride synthesis, gluconeogenesis, p38MAPK cascade regulation, cholesterol biosynthesis (Polish-Red versus Hereford); IGF-receptor signaling, catecholamine transport, lipoprotein lipase, tyrosine kinase binding receptor (Polish-HF versus Hereford), and PGF-receptor binding, (Polish-HF versus Polish-Red). Validation results showed that the relative expression values were consistent to those obtained by RNA-seq, and significantly correlated between the quantitative reverse transcription PCR (RT-qPCR) and RNA-seq (Pearson's r > 0.90). Our results provide new insights on bovine liver gene expressions among dairy versus dual versus beef breeds by identifying the large numbers of DEGs markers submitted to NCBI gene expression omnibus (GEO) accession number GSE114233, which can serve as useful genetic tools to develop the gene assays for trait-associated studies as well as, to effectively implement in genomics selection (GS) cattle breeding programs in Poland.

A porcine gluteus medius muscle genome-wide transcriptome analysis: dietary effects of omega-6 and omega-3 fatty acids on biological mechanisms.

BACKGROUND: The level of omega-6 and omega-3 polyunsaturated fatty acids can affect many cellular systems and function via nuclear receptors or the bioactive lipid regulation of gene expression. The objective of this study was to investigate changes in the muscle transcriptome and the biological functions regulated by increased consumption of omega-3 and omega-6 fatty acids in the pig gluteus medius muscle. RESULTS: The transcriptome of the gluteus medius muscle was studied for pigs subjected to either a control diet or a diet supplemented with linseed and rapeseed oil to increase polyunsaturated fatty acid content. Next-generation sequencing (NGS) was used to generate the muscle tissue transcriptome database pointing differentially expressed genes (DEG). Comparative expression analyses identified 749 genes significantly differing at least in the twofold of change between two groups of animals fed with divergent level of omega-3 and omega-6 fatty acids. The expression of 219 genes was upregulated, and the expression of 530 genes was downregulated in the group of pigs supplemented with omega-3 and omega-6 fatty acids in relation to control group pigs. Results of RNA-seq indicated a role of fatty acid in the regulation of the expression of genes which are essential for muscle tissue development and functioning. Functional analysis revealed that the identified genes were important for a number of biological processes including inflammatory response, signaling, lipid metabolism, and homeostasis. CONCLUSIONS: Summarizing, obtained results provide strong evidence that omega-6 and omega-3 fatty acids regulate fundamental metabolic processes in muscle tissue development and functioning.

Effect of a diet enriched with omega-6 and omega-3 fatty acids on the pig liver transcriptome.

The optimal ratio of omega-6 to omega-3 polyunsaturated fatty acids (PUFAs) is important for keeping the homeostasis of biological processes and metabolism, yet the underlying biological mechanism is poorly understood. The objective of this study was to identify changes in the pig liver transcriptome induced by a diet enriched with omega-6 and omega-3 fatty acids and to characterize the biological mechanisms related to PUFA metabolism. Polish Landrace pigs (n = 12) were fed diet enriched with linoleic acid (LA, omega-6) and α-linolenic acid (ALA, omega-3) or standard diet as a control. The fatty acid profiling was assayed in order to verify how feeding influenced the fatty acid content in the liver, and subsequently next-generation sequencing (NGS) was used to identify differentially expressed genes (DEG) between transcriptomes between dietary groups. The biological mechanisms and pathway interaction networks were identified using DAVID and Cytoscape tools. Fatty acid profile analysis indicated a higher contribution of PUFAs in the liver for LA- and ALA-enriched diet group, particularly for the omega-3 fatty acid family, but not omega-6. Next-generation sequencing identified 3565 DEG, 1484 of which were induced and 2081 were suppressed by PUFA supplementation. A low ratio of omega-6/omega-3 fatty acids resulted in the modulation of fatty acid metabolism pathways and over-representation of genes involved in energy metabolism, signal transduction, and immune response pathways. In conclusion, a diet enriched with omega-6 and omega-3 fatty acids altered the transcriptomic profile of the pig liver and would influence animal health status.