1,721,089 research outputs found
Espressione genica di biomarcatori di neurotossicità in embrioni di Zebrafish
No full textLo scopo del presente studio è quello di indagare, su di embrioni di zebrafish, gli effetti dei contaminanti ambientali organofosfati e carbammati. Per questo scopo su pool di larve si è valutata, tramite tecnica PCR Real Time (qPCR), l'espressione genica di alcuni geni geni coinvolti in diversi pathways che includono quello colinergico (AChE), il recettore NMDA (GRIN-1B), la differenziazione e mielinizzazione degli oligodendrociti (LINGO-1B) e l’attivazione neuronale generale (c-Fos). L’analisi è stata condotta in duplicato sia per i geni di interesse (Ache; GRIN1B; LINGO-1B e c-Fos) sia per I geni reference (beta actina-bACT; E74-like ETS transcription factor 1-ELF1; ribosomal protein L18-RPL18)
Characterization of in vitro model based on primary culture of human mammary epithelial cells (hMECs).
No full textThis data set contains results related to the characterization of an appropriate in vitro model based on human mammary epithelial cells (hMECs). The objective of the research was to verify the accuracy of hMECs as solid translational model for the study of mammary epithelial barrier. The results showed the characterization (morphological data; epithelial markers Epithelial-Cadherin (E-Cad) and Cytokeratin 18 (CK18); gene expression of drugs transporters and doubling Time (DT)), maintaining of hMEC primary culture. The hMECs maintained until P6 showed a typical cobblestone morphology and doubling time data showed a mean value of 36.6 ± 3.25 hours (see data file CONCEPTION_WP3_hMECs_DoublingTime_20240723.csv). The hMEC expressed epithelial markers Epithelial-Cadherin (E-Cad) and Cytokeratin 18 (CK18), confirming their epithelial origin (see data file CONCEPTION_WP3_hMECs_FlowCytometer_20240723.csv). In addition we analyze the gene expression of 84 drugs transporters (by using a commercial kit, RT2) and the Ct value was reported in CONCEPTION_WP3_hMECs_RT2ARRAY_20240723.xlsx) The transcriptional profile of drug transporters showed a differential level of gene expression ranging from ΔCt values (Ct mean reference genes – Ct interest gene) very negative (lower expression) to ΔCt values less negative (higher expression). Only 3 were not detectable (SLC22A9, SCCO1B3 and SLCO1B3). Collectively, the results show that primary culture of hMECs express epithelial cell markers and tight junctions molecule and express the main drug transporters, suggesting that it will be used to study epithelial barrier functions
ConcePTION WP3 task 3.2. Isolation and characterization of Mammary Epithelial Cells from Göttingen minipig (mpMECs): an in vitro study to compare mpMECs vs pMECs (porcine Mammary Epithelial Cells)
No full textThis data set contains data related to the characterization of an appropriate in vitro animal model based on primary culture of Göttingen Minipig Mammary Epithelial Cells (mpMECs). The objective of the research was to verify the accuracy of mpMECs as solid translational model for the study of mammary epithelial barrier and compare mpMECs results with those obtained from Mammary Epithelial Cells isolated from hybrid commercial pig (pMECs). The results showed that it was possible to isolate, culture and expand three pure epithelial cell lines obtained from three different animals. The mpMECs maintained until P10 showed a typical cobblestone morphology and a similar doubling time profile (MG9 32.6 ± 4 h, MG11 30.5 ± 3 h and MG12 27.4 ± 2.8 h, as reported in the data file CONCEPTION_WP3_mpMECs_DoublingTime_09032023.xlsx). DNA index was normal for all the three cell populations with a mean value of 0.98 ± 0.01. Regarding the cell cycle, the cell populations MG9, MG11 and MG12 showed the three distinct phases that could be recognized in a proliferating cell population: G0/G1, S and G2/M (see data file CONCEPTION_WP3_mpMECs_CellCycle_DNAIndex_09032023.xlsx). All the three mpMECs cell lines expressed epithelial markers Epithelial-Cadherin (E-Cad) and Cytokeratin 18 (CK18), confirming their epithelial origin (see data file CONCEPTION_WP3_mpMECs_FlowCytometer_E-Cad_CK18_09032023.xlsx). In particular in MG12, the contour of CK18 positive peak showed a shoulder suggesting the presence of a cellular subpopulation with a particularly high positivity. The barrier function of mpMECs was evaluated via TEER and fluorescein sodium (SF) transport, the formation of the monolayer integrity was evaluated in all the three primary mpMECs and in the pre-mixed pool of mpMECs and pMECs. MG9, MG11 and MG12 resulted in a similar TEER profile at the 0.15×10^6 cells, achieving higher TEER and lower SF values at the day 3 and 4 of culture, which was also confirmed in the pre-mixed pool case. Moreover, by comparing the maximum values of TEER reached at the different seeding density tested, a significative difference resulted in MG9 with respect to MG11, MG12 and pool. From a similar comparison with minimum values of SF transport, no difference between the groups was showed. Finally, in pre-mixed pools of mpMECs and pMECs a difference in the ability to form the epithelial barrier was shown: mpMEC barrier resulted less tight compact then that formed by pMECs. (see data file CONCEPTION_WP3_mpMECs_TEER_SF_09032023.xlsx). On pre-mixed pool of mpMECs and pMECs we evaluated the bioenergetic metabolism: no difference in energy production under basal cell culture condition was observed but under stressed state pMECs made more efficient use of mitochondrial oxidative metabolism than mpMECs, conversely, these last ones could more efficiently increased the glycolytic activity (see data file CONCEPTION_WP3_mpMECs_BioenergerticMetabolism_09032023.xlsx). The transcriptional profile of drug transporters evaluated in pre-mixed pool of mpMECs or pMECs cultured MECGM medium. Among the 84 genes, 66 genes were detectable, 18 genes were not detectable or higher than 35 threshold cycle, so considered as negative according to the handbook, in both cell lines.
No difference between mpMECs and pMECs drug transporter gene expression levels was observed (see data file CONCEPTION_WP3_mpMECs_RT2ARRAY_09032023.xlsx)
ConcePTION WP3 task 3.2. Isolation of porcine Mammary Epithelial Cells (pMECs). In vitro model for epithelial barrier
No full textThe present research aimed to develop a method for the isolation and culture of porcine Mammary Epithelial Cells (pMECs) to study mammary epithelial barrier in vitro. In agreement with the application of the 3Rs, porcine mammary gland tissues were collected at a local slaughterhouse and, after dissociation, the cells were isolated, cultured and characterized by morphology, cell cycle analysis and immunophenotyping. The ability to create a barrier was tested by TEER (Transepithelial/transendothelial electrical resistance) and the gene expression of genes related to drug transporters was evaluated by a PCR array. All the three pMECs cellular lines isolated are able to create a tight barrier and express the main ABC and SLC drug transporters confirming that the method of isolation is efficient to obtain primary pMECs lines to study epithelial barrier functions in the pig model. This activity was carried out as a part of Conception project (GA 821520)
Phenotypic parameters, gene expression and calprotectin level in high salt‑fed stroke‑prone spontaneously hypertensive rats
No full textThis dataset contains data for a study in which a high-salt/low-potassium stroke permissive diet (Japanese Diet; JD) was fed to Spontaneously Hypertensive Stroke Prone Rats (SHRSPs) and to Spontaneously Hypertensive Stroke Resistant Rats (SHRSRs). A Regular Diet (RD) was used as control diet. Specifically, for SHRSP and SHRSR rats fed with JD or RD are reported: (1) phenotypic parameters data (body weight, proteinuria and blood pressure), (2) perivascular and glomerular fibrosis (% area) data, (3) ZO-1, Ocln, Actb, GAPDH and Pgk1 gene expression data in the small intestine, (4) data about serum calprotectin levels.
We examined the inflammatory status of the animals by measuring serum calprotectin levels, and the gut barrier integrity by assessing gene expression of the tight-junction proteins zonulin (ZO-1) and occludin (Ocln) after 4 weeks of diet (short-term; ST) or until stroke occurrence for a maximum of 10 weeks (long-term; LT). Phenotypic parameters as body weight (BW), proteinuria, blood pressure (BP) and perivascular-glomerular fibrosis (% area) were measured. The BW of the two different strains fed RD increase with a similar trend, while the BW growth of SHRSP fed JD is significantly lower than SHRSR from the 5th week of diet. After 4 weeks, proteinuria level was significantly increased in SHRSPs JD-ST compared to RD-fed SHRSPs, and continued to increase throughout JD administration. Systolic blood pressure (BP) was not different among the four groups after 4 week of diet but increased significantly over time in both SHRSRs JD-LT and SHRSPs JD-LT (see file SHRSP_JapaneseDiet_PhenotypicParameters_23082024.csv). Perivascular and glomerular fibrosis significantly increased in SHRSPs, but not SHRSRs, fed JD for 4 weeks compared with the corresponding controls fed RD (see file SHRSP_JapaneseDiet_Fibrosis_23082024.csv).
Although SHRSPs carry a vasculitis-like cerebrovascular damage, no strain/diet-related significant differences in serum calprotectin levels were detected among experimental groups (see file SHRSP_JapaneseDiet_SerumCalprotectin_23082024.csv). For the gene expression of the tight-junction proteins all samples were analyzed in duplicate. RT-qPCR assays were carried out for the target genes ZO-1 and Ocln and three different reference genes, Actb (Actin beta), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and Pgk1 (Phosphoglycerate kinase 1). The duplicates were averaged, then ZO-1 and Ocln mRNA data were normalized based on the geometric mean of the Ct of the three reference genes (ΔCt = Ctmean ref. genes – Ctinterest gene). For each strain, the relative gene expression of the studied genes in JD-fed animals was calculated as fold change using the 2−ΔΔCt method in relation to the RD T0 time point (ΔΔCt = ΔCtT0 JD or T1 JD–ΔCtT0 RD). Undetectable RT-qPCR Cts were assigned a value of 40 to avoid overestimation of means.
In our study, ZO-1 expression did not change between the two rat strains on the two diets. In contrast, Ocln expression was significantly lower in SHRSPs compared to SHRSRs upon RD and increased in a time-dependent manner only in JD-fed SHRSPs, suggesting a strain-dependent interaction with the diet (see file SHRSP_JapaneseDiet_GeneExpression_23082024.csv)
Expression of HSP70/HSC70 in swine blastocysts: Effects of oxidative and thermal stress.
No full textEffects of holding and the addition of naloxone on vitrification of equine immature oocytes
Full text linkThis study investigates the effects of overnight holding and naloxone (Nx) supplementation on the vitrification
outcomes of equine immature oocytes. Oocytes were divided into six experimental groups based on treatment
combinations: fresh (F) and held (H) control oocytes, oocytes vitrified with or without Nx (10 8 M) (VIT and VIT-
Nx), oocytes vitrified after overnight holding with or without Nx (10 8 M) (H-VIT and H-VIT-Nx). They were
assessed for survival, meiotic competence, intracellular oxidative stress, mitochondrial activity and distribution,
apoptosis, and apoptotic gene expression. At survival rate determination, the degeneration rate was higher in VIT
and VIT-Nx compared to F (P < 0.05). The highest maturation rate was observed in VIT-Nx. A significant
reduction in ROS levels was observed in H compared to F (P < 0.05). ROS levels were similar between F and VIT,
while the Nx supplementation tended to increase them (VIT-Nx vs F: P = 0.053; VIT-Nx vs VIT: P = 0.069).
Conversely, in oocytes vitrified after overnight holding, vitrification induced an increase in ROS levels (H vs VIT:
P < 0.05), which was not observed in H-VIT-Nx. GSH intracellular levels showed significant differences only in
held oocytes, with higher GH levels in H compared to H-VIT and H-VIT-Nx (P < 0.05). All treatments induced an
increase in HMMP levels compared to F (P < 0.05). In H oocytes, mitochondria were distributed throughout the
entire oolemma (TOMM20) and active mitochondria (D-LAT) were detected in the outermost region. Incontrast,
in H-VIT-Nx, potentially active mitochondria were spread throughout the cytoplasm. AnnexinV/PI staining
revealed that the percentage of viable oocytes was higher (P < 0.05) in F and H than in all vitrified/warmed
oocytes, and H-VIT-Nx had the highest degeneration rate (P < 0.05). RT-PCR analysis confirmed the detection for
both reference genes, and target genes BCL2 and Survivin in all samples. In contrast, BAX and p53 transcripts
were consistently undetectable. No significant differences were observed in the expression of BCL2 and Survivin
between groups. In conclusion, overnight holding at uncontrolled room temperature can alter oocyte charac-
teristics and lead to variable results after vitrification. Nx demonstrated contrasting antioxidant effects
depending on the vitrification timing, but it appeared to improve IVM outcomes in oocytes vitrified immediately
after collection
X and Y chromosome-bearing spermatozoa are equally able to uptake and internalize exogenous DNA by sperm-mediated gene transfer in swine
No full textSince proteomic differences between male X/Y chromosome-bearing gametes have recently been described, a question has been raised: could these differences be responsible for different behavior between X and Y chromosome-bearing spermatozoa during the binding and internalization of exogenous DNA in the swine species? In order to investigate this hypothesis, our group studied the process of the uptake and internalization of exogenous DNA in X and Y chromosome-bearing sperm sub-populations. No significant differences were found between sperm types in both the uptake and internalization of exogenous DNA. The quantity of internalized exogenous DNA was significantly lower than that of the uptaken DNA. In conclusion, our results showed that X and Y chromosomes-bearing spermatozoa have the same binding capacity and internalization of DNA, and the proteomic differences between them do not seem to interfere with these complex processe
Attività antinfiammatoria di un mangime complementare addizionato con estratto secco di Boswellia serrata e Salix alba in galline ovaiole
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