Pig Liver Tissue Research Articles

Assessment of the efficacy of a grape seed waste in counteracting the changes induced by aflatoxin B1 contaminated diet on performance, plasma, liver and intestinal tissues of pigs after weaning

The aim of this study was to investigate the potential of a grape seed byproduct to mitigate the harmful damage produced by aflatoxin B1 (AFB1) at systemic level in plasma and liver as well as at local level in the gastrointestinal tract in weaned piglets. Twenty four crossbred pigs (TOPIG) were randomly assigned to one of four experimental diets: 1)- control diet (normal compound feed for starter piglets without mycotoxin), 2)- AFB1 diet (compound feed contaminated with 320 ppb pure AFB1), 3)- GS diet (compound feed including 8% of grape seed meal), 4)- AFB1+GS diet (compound feed containing 8% of grape seed meal contaminated with 320 ppb AFB1) for 30 days. The results showed that pigs fed AFB1 diet had altered performance (−25.1%), increased the thiobarbituric substances (TBARS) concentration wile reduced total antioxidant capacity and activity of antioxidant enzymes (CAT, SOD and GPx) in plasma and organs. AFB1 produced a dual effect on inflammatory response by increasing the level of pro-inflammatory cytokines in liver and colon and decreasing these cytokines in duodenum. The inclusion of grape seed in the diet of AFB1 intoxicated pigs enhanced the antioxidant enzymes activity, decreased the pro-inflammatory cytokines and TBARS level and ameliorated the growth performance of AFB1-treated animals. These findings suggest that grape waste is a promising feed source in counteracting the harmful effect of aflatoxin B1.

GASP and FASP are Complementary for LC–MS/MS Proteomic Analysis of Drug‐Metabolizing Enzymes and Transporters in Pig Liver

Sample preparation is a critical step in the proteomic workflow. Numerous different approaches are used, tailored to the type of sample, the aims of the experiment, analytical method, and to an extent, user preference. This has resulted in large variation in reported protein abundances. In this study, the complementarity of two different sample preparation techniques is demonstrated for the study of absorption, distribution, metabolism, and excretion (ADME) related proteins from pig liver tissue. Filter-aided sample preparation (FASP) is a well-established and widely used method, while gel-aided sample preparation (GASP) is a relatively new method optimized and simplified from previous gel-associated digestion techniques. To investigate each method, the number of peptides and proteins characterized, reproducibility of results, and their real-time application are examined. While both methods have their merits and limitations, for example, FASP is the less technical of the two methods, while GASP is time efficient, ultimately the two methods show significant differences in the peptides identified and therefore, the use of both methods should be considered when examining and quantifying ADME related proteins. Data are available via ProteomeXchange with identifier PXD011324.

Effects of enrofloxacin on antioxidant system, microsomal enzymatic activity, and proteomics in porcine liver.

Enrofloxacin (EF) is a widely used fluoroquinolone, usually regarded as a safe and effective treatment for bacterial infections. Adverse effects of EF have previously been demonstrated in some species, but so far there have been no studies looking specifically at the impact of EF on pigs. In this study, three different doses of EF (5, 25 and 125mgkgbw-1 ) were administrated to Bama pigs. The results showed that lipid peroxidation of pig liver tissue occurred with all EF doses. The 125mgkg dose of EF induced catalase (CAT) and glutathione peroxidase (GSH-px) and increased CYP450 content in pig liver microsomes. The activity of microsomal NADPH-cytochrome C reductase (NCCR) was elevated at both the 25 and 125mgkg doses of EF. Microsomal erythromycin N-demethylase (ERND) and aminopyrin N-demethylase (AND) were inhibited by high doses of EF, while aniline-4-hydroxylase (AH) was unaffected. None of the EF treatments affected superoxide dismutase (SOD) or cytochrome b5 content. Antioxidases and microsomal enzymes may work together to resist the adverse effects of EF. Proteomic analysis revealed increased protein expression of carboxylesterase (CES) and alpha-enolase (ENO1) in microsomes as a stress response to EF. These results provide new information about the adverse effect of fluoroquinolones and help guide their usage more effectively in the clinic or animal breeding.

Transglycosylated Starch Modulates the Gut Microbiome and Expression of Genes Related to Lipid Synthesis in Liver and Adipose Tissue of Pigs

Dietary inclusion of resistant starches can promote host health through modulation of the gastrointestinal microbiota, short-chain fatty acid (SCFA) profiles, and lipid metabolism. This study investigated the impact of a transglycosylated cornstarch (TGS) on gastric, ileal, cecal, proximal-colonic, and mid-colonic bacterial community profiles and fermentation metabolites using a growing pig model. It additionally evaluated the effect of TGS on the expression of host genes related to glucose and SCFA absorption, incretins, and satiety in the gut as well as host genes related to lipid metabolism in hepatic and adipose tissue. Sixteen growing pigs (4 months of age) were fed either a TGS or control (CON) diet for 11 days. Bacterial profiles were determined via Illumina MiSeq sequencing of the V3–5 region of the 16S rRNA gene, whereas SCFA and gene expression were measured using gas chromatography and reverse transcription-quantitative PCR. Megasphaera, which was increased at all gut sites, began to benefit from TGS feeding in gastric digesta, likely through cross-feeding with other microbes, such as Lactobacillus. Shifts in the bacterial profiles from dietary TGS consumption in the cecum, proximal colon, and mid colon were similar. Relative abundances of Ruminococcus and unclassified Ruminococcaceae genus were lower, whereas that of unclassified Veillonellaceae genus was higher in TGS- compared to CON-fed pigs (p < 0.05). TGS consumption also increased (p < 0.05) concentrations of SCFA, especially propionate, and lactate in the distal hindgut compared to the CON diet which might have up-regulated GLP1 expression in the cecum (p < 0.05) and mid colon compared to the control diet (p < 0.10). TGS-fed pigs showed increased hepatic and decreased adipocyte expression of genes for lipid synthesis (FASN, SREBP1, and ACACA) compared to CON-fed pigs, which may be related to postprandial portal nutrient flow and reduced systemic insulin signaling. Overall, our data show that TGS consumption may affect gastrointestinal bacterial signaling, caused by changes in gut bacterial profiles and the action of propionate, and host lipid metabolism.

Squalene epoxidase plays a critical role in determining pig meat quality by regulating adipogenesis, myogenesis, and ROS scavengers

In mammals, Squalene epoxidase (SQLE) is an enzyme that converts squalene to 2,3-oxidosqualene, in the early stage of cholesterol generation. Here, we identified single nucleotide polymorphisms (SNPs) in the SQLE gene (c.2565 G > T) by RNA Sequencing from the liver tissue of Berkshire pigs. Furthermore, we found that homozygous GG pigs expressed more SQLE mRNA than GT heterozygous and TT homozygous pigs in longissimus dorsi tissue. Next, we showed that the SNP in the SQLE gene was associated with several meat quality traits including backfat thickness, carcass weight, meat colour (yellowness), fat composition, and water-holding capacity. Rates of myogenesis and adipogenesis induced in C2C12 cells and 3T3-L1 cells, respectively, were decreased by Sqle knockdown. Additionally, the expression of myogenic marker genes (Myog, Myod, and Myh4) and adipogenic marker genes (Pparg, Cebpa, and Adipoq) was substantially downregulated in cells transfected with Sqle siRNA. Moreover, mRNA expression levels of ROS scavengers, which affect meat quality by altering protein oxidation processes, were significantly downregulated by Sqle knockdown. Taken together, our results suggest the molecular mechanism by which SNPs in the SQLE gene can affect meat quality.

Validation of a mathematical model for laser-induced thermotherapy in liver tissue.

The purpose of the study was to develop a simulation approach for laser-induced thermotherapy (LITT) that is based on mathematical models for radiation transport, heat transport, and tissue damage. The LITT ablation was applied to ex vivo pig liver tissue. Experiments were repeated with different laser powers, i.e., 22-34 W, and flow rates of the cooling water in the applicator system, i.e., 47-92 ml/min. During the procedure, the temperature was measured in the liver sample at different distances to the applicator as well as in the cooling circuit using a fiber optic thermometer. For validation, the simulation results were compared with the results of the laser ablation experiments in the ex vivo pig liver samples. The simulated and measured temperature curves presented a relatively good agreement. The Bland-Altman plot showed an average of temperature differences of -0.13 ∘C and 95%-limits-of-agreement of ±7.11 ∘C. The standard deviation amounted to ±3.63 ∘C. The accuracy of the developed simulation is comparable with the accuracy of the MR thermometry reported in other clinical studies. The simulation showed a significant potential for the application in treatment planning.

Protective and Heat Retention Effects of Thermo-sensitive Basement Membrane Extract (Matrigel) in Hepatic Radiofrequency Ablation in an Experimental Animal Study.

To evaluate the protective effect of using thermo-sensitive basement membrane extract (Matrigel) for hydrodissection to minimize thermal injury to nearby structures and to evaluate its heat sink effect on the ablation zone in radiofrequency ablation (RFA) of the liver. First, the viscosity profile and heat sink effect of Matrigel were assessed during RFA in vitro and ex vivo. Fresh pig liver tissue was used, and the temperature changes in Matrigel and in 5% dextrose in water (D5W) during RFA were recorded. Then, the size of the ablation zone in the peripheral liver after RFA was measured. Second, in an in vivo study, 45 Sprague-Dawley rats were divided into three groups of 15 rats each (Matrigel, D5W and control). In the experimental groups, artificial ascites with 10ml of Matrigel or D5W were injected using ultrasound guidance prior to RFA. The frequency of thermal injury to the nearby organs was compared among the three groups, with assessments of several locations: near the diaphragm, the abdominal wall and the gastrointestinal (GI) tract. Finally, the biological degradation of Matrigel by ultrasound was evaluated over 60days. First, Matrigel produced a greater heat retention (less heat sink) effect than D5W during ex vivo ablation (63±9 vs. 26±6°C at 1min on the surface of the liver, P<0.001). Hepatic ablation zone volume did not differ between the two groups. Second, thermal injury to the nearby structures was found in 14 of 15 cases (93.3%) in the control group, 8 of 15 cases (53.3%) in the D5W group, and 1 of 15 cases (6.7%) in the Matrigel group. Significant differences in the thermal injury rates for nearby structures were detected among the three groups (P<0.001). The most significant difference in the thermal injury rate was found in locations near the GI tract (P=0.003). Finally, Matrigel that was injected in vivo was gradually degraded during the following 60days. Using thermo-sensitive Matrigel as a hydrodissection material might help reduce the frequency of collateral thermal injury to nearby structures, especially in locations close to the GI tract, compared to conventional D5W. Additionally, Matrigel did not increase the heat sink effect on the ablation zone during ablation and was degraded over time in vivo.

The association of haplotypes in IGFBP-3 gene promoter region and tissue expressions in three pig breeds

ABSTRACTBama Xiang pig (BM) and Tibetan mini-pig (TM) are used as experimental animals in China; however, the dwarf molecular mechanisms of these Chinese local pig breeds are unknown. IGFBP-3 affects animal growth, carcass and meat quality. The aim of this study was to identify the polymorphisms in the promoter of the IGFBP-3 and analyse their effect on the IGFBP-3 mRNA expression level in liver and muscle tissues. High-density single-nucleotide polymorphisms (SNPs) (31) and InDels (5) were detected in the promoter of the IGFBP-3 in the BM, TM and Junmu No. 1 White (JM, control) pig breeds from 114 individuals by re-sequencing. A perfect Linkage disequilibrium consisted of 13 SNPs was observed in the promoter region and 2 main haplotypes were identified, of which the h1 genotype (GCA-ATGTACATAT) was more prevalent in JM breed than in TM or BM breeds (P < .0001), h2 (ATGTGCACG--CGC) was the dominant haplotype in TM and BM breeds (P < .0001). Expression analysis showed that haplotype of the promoter region is highly associated with the IGFBP-3 mRNA expression level in liver and muscle tissues of pigs. The IGFBP-3 mRNA expression level was determined higher in the liver and muscle tissues of pigs with h2 genotype as compared to that in pigs with h1 genotype (P < .05). The results suggest that the SNPs and haplotypes in the promoter of the IGFBP-3 gene may serve as useful molecular markers for the body growth traits and the breeding in swine.

Esterification of essential and non-essential fatty acids into distinct lipid classes in ruminant and non-ruminant tissues.

Extensive microbial biohydrogenation of polyunsaturated fatty acids (PUFA) in the rumen reduces the essential fatty acids (EFA) available for absorption in ruminant animals, but there is no published documentation of ruminants developing EFA deficiency. In ruminants, most circulating PUFA are found in the phospholipid (PL) and cholesteryl ester lipid classes that have slow turn-over compared to other lipid classes. The objective of this experiment was to measure fatty acid esterification patterns of the non-EFA palmitic (16:0) and oleic acid (18:1), and the EFA linoleic (18:2) and linolenic acid (18:3) in small intestine, liver, and muscle tissue of cows and pigs to identify tissues participating in sequestration of these FA in less metabolically active lipid classes in ruminants. Bovine and porcine small intestine, liver, and muscle explants were prepared and incubated in media containing radiolabeled 16:0, 18:1, 18:2, or 18:3 to measure esterification of fatty acids into PL and TG. Neither bovine nor porcine small intestine explants preferentially incorporated non-EFA compared to EFA into PL vs TG. Bovine liver explants esterified a larger proportion of EFA than non-EFA into PL compared to TG, while incorporation was similar among the FA tested in porcine liver explants. Bovine muscle explants showed preferential incorporation of EFA into PL rather than TG. Results show that bovine and porcine liver and muscle esterify EFA and non-EFA differently and that the conservation of EFA in ruminants is a result of preferential incorporation of EFA into PL mediated by bovine liver and muscle, but not the small intestine.

Analysis of electrocautery generated smoke by chromatographic-mass spectrometry.

to analyze the chemical components of the smoke from electrocautery from coagulating muscle and liver tissues of pigs. we collected smoke produced by electrocautery applied to porcine tissue in previously evacuated bottles, with qualitative and quantitative analysis of the compounds present through the hyphenated technique gas chromatography / mass spectrometry. there was a majority of decanal aldehyde in the fumes from the subcutaneous, muscle and liver tissues. Fumes of subcutaneous and muscular tissues also showed the presence of hexanal and phenol. In the fumes of subcutaneous and liver tissues we also found toluene and limonene and, finally, nonanal smoke was present in the muscle and liver tissues. there is increasing evidence showing that smoke from electrocautery used in subcutaneous, muscle and liver tissue is harmful to human health. Thus, there is need to reduce exposure to it or wear masks with filters capable of retaining these particles. analisar quimicamente os componentes da fumaça do eletrocautério, provenientes da coagulação de tecidos, muscular e hepático de suino. coleta de fumaça produzida por eletrocauterização de tecido porcino em frascos previamente evacuados com análise qualitativa e quantitativa dos compostos presentes, através de técnica hifenada, cromatografia a gás/espectrometria de massas. houve presença majoritária do aldeído decanal nas fumaças provenientes dos tecidos subcutâneo, muscular e hepático. Fumaças dos tecidos subcutâneo e muscular mostraram também a presença de hexanal e fenol. Nas fumaças dos tecidos subcutâneo e hepático foram encontrados ainda tolueno e limoneno e, por fim, nonanal estava presente nas fumaças dos tecidos muscular e hepático. há número crescente de evidências mostrando que fumaça proveniente de eletrocauterização de tecidos subcutâneo, muscular e hepático é nociva à saúde de seres humanos. Portanto, há necessidade de reduzir a exposição a ela ou usar máscara com filtro capaz de reter essas partículas.

Alcohol modulates autophagy and apoptosis in pig liver tissue

BackgroundAutophagy serves as a cellular protective mechanism against alcohol-induced tissue injury but excessive autophagy can also be detrimental leading to apoptosis. Our laboratory has previously shown that moderate alcohol consumption alters expression of proteins in the insulin signaling pathway and worsens glucose metabolism in the liver in a swine model of metabolic syndrome. We examined the effect of alcohol consumption on apoptosis and autophagy signaling in the liver in our clinically relevant animal model of chronic hypercholesterolemia. Material and methodsTwenty-six Yorkshire swine were fed a high-fat diet for 4 wks and were then split into three groups: hypercholesterolemic diet alone (HCC, n = 9), hypercholesterolemic diet with vodka (hypercholesterolemic vodka [HCV], n = 9), and hypercholesterolemic diet with wine (hypercholesterolemic wine [HCW], n = 8) for 7 wks. Animals underwent euthanasia, and liver tissue samples were harvested for analysis. Liver tissue was analyzed via Western blot analysis. Protein density data were normalized to GAPDH and is reported as fold-change values ± standard error of the mean compared to the high-cholesterol diet control group. A Kruskal–Wallis test with a Dunn's multiple comparison test was used to compare the means among groups. ResultsThe HCV group showed significant increases in several proapoptotic proteins (including caspase 3, caspase 8, caspase 9, and cleaved caspase 9) compared with the HCC group. There was a decrease in the proapoptotic protein (BAD) and an increase in anti-apoptotic signal (B-cell lymphoma-2) in the HCW group compared with HCC control. There were increases in pro-survival proteins (AKT, p-AKT, mTOR, p-mTOR) in the HCW and the HCV group compared with control (HCC). There were decreases in autophagy protein LCB-3 in the HCW and HCV compared with the control. ConclusionsWe found that moderate alcohol consumption altered protein expression related to apoptosis and autophagy signaling in pig liver in the setting of hypercholesterolemia. Interestingly, vodka may induce proapoptotic pathways in liver tissue, whereas wine may induce anti-apoptotic signaling. These results provide a mechanism by which vodka may contribute to alcoholic liver disease and supports the notion that wine, containing resveratrol, may prevent cellular apoptosis in liver tissue in the setting of hypercholesterolemia.

Exploration of steroidogenesis-related genes in testes, ovaries, adrenals, liver and adipose tissue in pigs.

To explore the metabolism of steroids in the pig species, a qualitative PCR analysis was performed for the main transcript of 27 genes involved in steroid metabolism. We compared samples of testes, adipose tissue and liver from immature and peripubertal males, adrenal cortex from peripubertal males, ovaries from cyclic females and adipose tissue from peripubertal females. Some genes were shown to have a tissue-specific expression. Two of them were expressed only in testes, ovaries and adrenals: CYP11A1 and CYP11B. The CYP21 and HSD17B3 genes, were expressed respectively only in adrenals and only in testes. Very few differences were observed between transcriptional patterns of peripubertal testes and adrenal glands as well as between male and female fat tissues. However, the expression of genes involved in the sulfonation of steroids was higher in testes than in adrenals from males. Main differences between ovaries and testes were observed for HSD17B1/2/3, AKR1C-pig6 and sulfotransferase genes (SULT2A1/SULT2B1). The present study shows that the SRD5A2 and CYP21 genes were not involved in the testicular biosynthesis of androstenone. It also shows that porcine adrenal glands produce essentially corticosteroids and that fat tissue is unable to produce de novo steroids.

Method for Estimating the Acoustic Pressure in Tissues Using Low-Amplitude Measurements in Water

The aim of this study was to evaluate a simple, reliable and reproducible method for accuracy in estimating the acoustic pressure delivered in tissue exposed to ultrasound. Such a method would be useful for therapeutic applications of ultrasound with microbubbles, for example, sonoporation. The method is based on (i) low-amplitude water measurements that are easily made and do not suffer from non-linear propagation effects, and (ii) the attenuation coefficient of the tissue of interest. The range of validity of the extrapolation method for different attenuation and pressure values was evaluated with a non-linear propagation theoretical model. Depending on the specific tissue attenuation, the method produces good estimates of pressures in excess of 10 MPa. Ex vivo machine-perfused pig liver tissue was used to validate the method for source pressures up to 3.5 MPa. The method can be used to estimate the delivered pressure in vivo in diagnostic and therapeutic applications of ultrasound.

Changes in the Intestinal Microbiota and Host Inflammatory Gene Expression in Pigs Fed a Flavanol‐Enriched Cocoa Powder

To determine the effects of eating cocoa‐derived flavanols on the composition of the gut microbiota, associated metabolic profiles, and related features of host intestinal health, pigs were fed with 0, 2.5, 10, or 20 grams of flavanol‐enriched cocoa powder/day for 27 days. Analysis of phase II metabolites of cocoa‐derived flavanols demonstrated that O‐methyl‐(epi)catechin‐glucuronide conjugates were detected in a dose‐dependent manner in urine, serum, liver, and adipose tissue of pigs fed cocoa powder. Products of microbial metabolism such as phenylpropionic acids, phenylacetic acids, benzoic acids, cinnamic acids, and conjugated benzoic acids were detected in most of the tissue and bio‐fluids examined. Cocoa consumption also changed the composition of the intestinal microbiome. Pigs fed 20 gram of cocoa/day had significantly increased abundance of Lactobacillus species (casei group) in the feces and distal colon contents compared to pigs not fed cocoa. Moreover, consumption of cocoa powder reduced the gene expression of tumor necrosis factor‐α and toll‐like receptors‐2, ‐4, and ‐9 in most intestinal tissues examined. Overall, this study demonstrated that consumption of flavanol‐enriched cocoa can contribute to gut health by enhancing the abundance of Lactobacillus species and modulating markers of localized intestinal immunity.

Heat Damage Zones Created by Different Energy Sources Used in the Treatment of Benign Prostatic Hyperplasia in a Pig Liver Model.

There are different types of transurethral prostatic surgeries and the complication profiles are different. This study aims to compare the heat damage zones (HDZ) created by five different technologies in a pig liver model. Monopolar resection, bipolar resection, electrovaporization, and Greenlight™ lasers of 120 and 180 W were used to remove fresh pig liver tissue in a simulated model. Each procedure was repeated in five specimens. Two blocks were selected from each specimen to measure the three deepest HDZ. The mean of HDZ was 295, 234, 192, 673, and 567 μm, respectively, for monopolar resection, bipolar resection, electrovaporization, Greenlight laser 120 W, and Greenlight laser 180 W, respectively. The Greenlight laser produced one to three times deeper HDZ than the other energy sources (p=0.000). Both 120 and 180 W Greenlight lasers produced deeper HDZ than the other energy sources. Urologists need to be aware of HDZ that cause tissue damage outside the operative field.

Single nucleotide polymorphism scanning and expression of the pig PPARGC1A gene in different breeds.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) is a candidate gene for lean meat production because it plays a key role in lipid metabolism. In this study, SNPs within the porcine PPARGC1A gene were investigated using PCR-sequencing and PCR-RFLP. Quantitative real-time PCR and Western blot were then used to analyze mRNA and protein expression in longissimus dorsi muscle (LM), liver, and backfat tissues of Dianna small-ear pigs (DSP, n = 6), Tibetan pigs (TP, n = 6), and large white pigs (LW, n = 6). Five novel SNPs (g.-1269A>G in the 5'-upstream regulatory region; g.190C>T, g.218C>A and g.234C>A in exon 8; and g.20C>T in intron 10) and three previously identified SNPs (g.417A>T in exon 8; g.56C>A in exon 9; and g.34G>A in intron 9) were found. Of these, only two, g.-1269A>G and g.234C>A, had three different genotypes in the three breeds (DSP, n = 63; TP, n = 51; and LW, n = 52). Expression was highest in LM, modest in the liver, and minimal in backfat. In LM tissue, LW had higher mRNA and protein levels than DSP and TP (P < 0.05), and there was a negative correlation between gene expression and intramuscular fat (IMF) content. LW had numerically higher expression in liver and backfat tissues than DSP and TP, and the differences in protein levels were significant (P < 0.05 in liver, P < 0.01 in backfat). In conclusion, PPARGC1A may play a key role in down-regulating lipid deposition, and the SNPs with differential genotype distribution among the three pig breeds may be related to gene expression and fat deposition.

Quantitative assessment of iron in heart and liver phantoms using dual-energy computed tomography.

The aim of the present study was to determine the correlation between dual-energy computed tomography (DECT) Hounsfield units (HU) and iron concentration, as well as the correlation between HU and magnetic resonance imaging (MRI)-derived R2* values, in phantoms of the heart and liver tissue. Phantoms were constructed containing pig heart or liver tissue and varying concentrations of iron (0.1, 5, 10, 15, 20 and 25 mg/ml). The phantoms were then examined by DECT and MRI. Linear regression analysis was used to determine the correlations between HU and iron concentration and HU and R2* values. The HU value of DECT increased with increasing iron concentrations in the liver and heart phantoms in a linear manner. The slope of the HU value change against iron concentration revealed that ΔH80–140 provided a better discernment of iron concentration as compared with ΔH100–140. The derived R2 values were all >0.9 for the associations of DECT and MRI measurements with iron concentrations. Therefore, DECT may be used for the determination of iron concentration in the liver and heart tissue, with the results correlating with those obtained with MRI.

Identification and characterization of RFRP gene in pigs and its association with reproductive traits

RFamide-related peptide (RFRP) gene is a crucial gene of the hypothalamo-pituitary-gonadal axis and plays an important role in reproduction regulation. This study aimed to clone and characterize the pig RFRP gene. We obtained a 645-bp cDNA of pig RFRP gene comprising a 546-bp open reading frame, which encoded a peptide of 188 amino acids. The pig RFRP coding sequences have the identities of 81, 68.8, and 76.1% with their counterparts in humans, mice, and rats, respectively. Real-time polymerase chain reaction showed that the pig RFRP gene was expressed predominantly in the stomach and testis of males, and large intestine and uterus of females. It was also found to be abundantly expressed in the cerebrum and liver tissues of both male and female pigs. The pig RFRP gene is 4.6 kb long and contains 3 exons and 2 introns. We further identified a total of 25 single nucleotide polymorphisms and 2 insertion/deletion (indel) polymorphisms across the whole RFRP gene, and 9 of them were in the exons. Marker trait association analysis in Yorkshire and Landrace populations showed that g.45859759C>T was significantly associated with the total number born of second parity (TBA2; P < 0.05). In conclusion, the pig RFRP gene was cloned and characterized in this study, and its polymorphism g.45859759C>T showed significant associations with reproductive traits.

Evaluation of YO-PRO-1 as an early marker of apoptosis following radiofrequency ablation of colon cancer liver metastases

Radiofrequency (RF) ablation (RFA) is a minimally invasive treatment for colorectal-cancer liver metastases (CLM) in selected nonsurgical patients. Unlike surgical resection, RFA is not followed by routine pathological examination of the target tumor and the surrounding liver tissue. The aim of this study was the evaluation of apoptotic events after RFA. Specifically, we evaluated YO-PRO-1 (YP1), a green fluorescent DNA marker for cells with compromised plasma membrane, as a potential, early marker of cell death. YP1 was applied on liver tissue adherent on the RF electrode used for CLM ablation, as well as on biopsy samples from the center and the margin of the ablation zone as depicted by dynamic CT immediately after RFA. Normal pig and mouse liver tissues were used for comparison. The same samples were also immunostained for fragmented DNA (TUNEL assay) and for active mitochondria (anti-OxPhos antibody). YP1 was also used simultaneously with propidium iodine (PI) to stain mouse liver and samples from ablated CLM. Following RFA of human CLM, more than 90% of cells were positive for YP1. In nonablated, dissected pig and mouse liver however, we found similar YP1 signals (93.1% and 65%, respectively). In samples of intact mouse liver parenchyma, there was a significantly smaller proportion of YP1 positive cells (22.7%). YP1 and PI staining was similar for ablated CLM. However in dissected normal mouse liver there was initial YP1 positivity and complete absence of the PI signal and only later there was PI signal. This is the first time that YP1 was applied in liver parenchymal tissue (rather than cell culture). The results suggest that YP1 is a very sensitive marker of early cellular events reflecting an early and widespread plasma membrane injury that allows YP1 penetration into the cells.

Prion protein (PrPc) interacts with histone H3 confirmed by affinity chromatography

The histones including H2a, H2b, H3 and H4 purified from pig liver tissue were immobilized onto Sepharose 4B to create a histone-Sepharose column. During chromatography of cow milk casein by histone-Sepharose column, two isoforms of prion protein (PrP(c)) with 34 and 30kDa molecular mass corresponding to diglycosylated and monoglycosylated PrP(c) respectively were found to be captured by histone ligands. To further verify the interaction between histones and PrP(c), the PrP(c)-Sepharose column was prepared and used to separate the histones. Two chromatography processes and SDS-PAGE demonstrated that only H3 in the histones was found to interact with PrP(c). This study suggested H3 could be the target molecule of PrP(C) in nuclei, which might be useful for understanding the prion disease.