Follicle-stimulating hormone regulates pro-apoptotic protein Bcl-2-interacting mediator of cell death-extra long (BimEL)-induced porcine granulosa cell apoptosis.

The pro-apoptotic protein Bim (B-cell lymphoma-2 (Bcl-2)-interacting modulator of cell death) has recently been identified and shown to promote cell death in response to several stimuli. In this report, we investigated the role of Bim in porcine follicular atresia. Initially, Bim cDNA was cloned and characterized from porcine ovarian tissue. Porcine Bim had three alternative splicing variants (Bim-extra long, Bim-long, and Bim-short), all containing the consensus Bcl-2 homology 3 domain. We then found the Bim-extra long (Bim(EL)) protein, the most abundant isoform of Bim, was strongly expressed and co-localized with apoptotic (TUNEL-positive) granulosa cells from porcine atretic follicles. Furthermore, overexpression of Bim(EL) triggered apoptosis in granulosa cells. In primary granulosa cell cultures under basal conditions, we observed that Bim(EL) expression was dampened by treatment with follicle-stimulating hormone (FSH). The role of the PI3K/Akt pathway in the regulation of repression was clarified by the use of the PI3K inhibitor, LY294002, and by transfection with Akt siRNA. Forkhead Box Protein O3a (FoxO3a), a well defined transcriptional activator of Bim, was phosphorylated at Ser-253 and inactivated after FSH stimulation. Also, FSH abolished FoxO3a nuclear accumulation in response to LY294002. Finally, chromatin immunoprecipitation assays demonstrated that FoxO3a directly bound and activated the bim promoter. Taken together, we conclude that Bim(EL) induces porcine granulosa cell apoptosis during follicular atresia, and its expression is regulated by FSH via the PI3K/Akt/FoxO3a pathway.

Oocyte-secreted growth differentiation factor 9 inhibits BCL-2-interacting mediator of cell death-extra long expression in porcine cumulus cell.

Oocyte-secreted factors (OSFs) maintain the low incidence of cumulus cell apoptosis. In this report, we described that the presence of oocytes suppressed the expression of proapoptotic protein BCL-2-interacting mediator of cell death-extra long (BIMEL) in porcine cumulus cells. Atretic (terminal deoxynucleotidyl transferase dUTP nick end labeling-positive) cumulus cells strongly expressed BIMEL protein. The healthy cumulus- oocyte complex exhibited a low BIMEL expression in cumulus cell while the removal of oocyte led to an about 2.5-fold (P < 0.5) increased expression in oocytectomized complex (OOX). Coculturing OOXs with denuded oocytes decreased BIMEL expression to the normal level. The similar expression pattern could also be achieved in OOXs treated with exogenous recombinant mouse growth differentiation factor 9 (GDF9), a well-characterized OSF. This inhibitory action of GDF9 was prevented by the addition of a phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Luciferase assay further demonstrated that BIM gene expression was forkhead box O3a (FOXO3a)-dependent because mutation of FOXO3a-binding site on the BIM promoter inhibited luciferase activities. Moreover, the activity of BIM promoter encompassing the FOXO3a-binding site could be regulated by GDF9. Additionally, we found that GDF9 elevated the levels of phosphorylated AKT and FOXO3a, and this process was independent of the SMAD signal pathway. Taken together, we concluded that OSFs, particularly GDF9, maintained the low level of BIMEL expression in cumulus cell through activation of the PI3K/FOXO3a pathway.

The disturbances of endoplasmic reticulum calcium homeostasis caused by increased intracellular reactive oxygen species contributes to fragmentation in aged porcine oocytes.

Accumulating evidence indicates that cellular and molecular abnormalities occur during oocyte aging, including fragmentation, increases in intracellular reactive oxygen species (ROS), and abnormal Ca(2+) oscillations. The objective of the present study was to characterize the relationships between intracellular ROS, Ca(2+) homeostasis of endoplasmic reticulum (ER), and fragmentation in aged porcine MII oocytes. Prolonged culture (36 h) of porcine oocytes resulted in elevated intracellular ROS level, impaired ER Ca(2+) homeostasis (i.e., Ca(2+) storage, Ca(2+) rising patterns after electroactivation, and the cluster distribution of ER), and increased fragmentation rates. However, when the porcine oocytes were treated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester), an intracellular Ca(2+) chelator, the fragmentation was significantly inhibited during in vitro aging. In order to pursue the underlying mechanisms, H2O2 and cycloheximide (CHX) were used to artificially increase or inhibit, respectively, the intracellular ROS levels in aged porcine oocytes during in vitro culture. The results demonstrated that incubation of porcine MII oocytes with H2O2 damaged the ER clusters and the Ca(2+) regulation of ER, leading to a high proportion of fragmented oocytes. In contrast, CHX, an intracellular inhibitor of ROS generation, prevented both increase of ROS level and damage of the ER Ca(2+) homeostasis in porcine oocytes during aging, resulting in low fragmentation rate. We conclude that the increased intracellular ROS damaged the ER clusters and ER Ca(2+) homeostasis, resulting in a disorder in ooplasmic free Ca(2+), which caused the fragmentations seen in porcine MII oocytes during aging.

MRI-Based Grading of Clear Cell Renal Cell Carcinoma Using a Machine Learning Classifier.

OBJECTIVE: To develop a machine learning (ML)-based classifier for discriminating between low-grade (ISUP I-II) and high-grade (ISUP III-IV) clear cell renal cell carcinomas (ccRCCs) using MRI textures. MATERIALS AND METHODS: We retrospectively evaluated a total of 99 patients (with 61 low-grade and 38 high-grade ccRCCs), who were randomly divided into a training set (n = 70) and a validation set (n = 29). Regions of interest (ROIs) of all tumors were manually drawn three times by a radiologist at the maximum lesion level of the cross-sectional CMP sequence images. The quantitative texture analysis software, MaZda, was used to extract texture features, including histograms, co-occurrence matrixes, run-length matrixes, gradient models, and autoregressive models. Reproducibility of the texture features was assessed with the intra-class correlation coefficient (ICC). Features were chosen based on their importance coefficients in a random forest model, while the multi-layer perceptron algorithm was used to build a classifier on the training set, which was later evaluated with the validation set. RESULTS: The ICCs of 257 texture features were equal to or higher than 0.80 (0.828-0.998. Six features, namely Kurtosis, 135dr_RLNonUni, Horzl_GLevNonU, 135dr_GLevNonU, S(4,4)Entropy, and S(0,5)SumEntrp, were chosen to develop the multi-layer perceptron classifier. A three-layer perceptron model, which has 229 nodes in the hidden layer, was trained on the training set. The accuracy of the model was 95.7% with the training set and 86.2% with the validation set. The areas under the receiver operating curves were 0.997 and 0.758 for the training and validation sets, respectively. CONCLUSIONS: A machine learning-based grading model was developed that can aid in the clinical diagnosis of clear cell renal cell carcinoma using MRI images.

[Application of the medical image three-dimensional visualization system of abdomen in diagnosis and evaluating resectability of pancreatic tumors].

OBJECTIVE: To study the value and the clinical application of the Medical Image three-dimensional Visualization System of Abdomen (MI-3DVS) in diagnosis and evaluating resectability of pancreatic tumor. METHODS: Twelve patients with pancreatic tumor were tested with 64-slice helical CT (64-MSCT) angiography, and the CT data was reconstructed with MI-3DVS from November 2008 to August 2009. The 3D findings were adopted in diagnosis and evaluating resectability, and the results were compared with surgical operation and the pathological finding. There were 7 male and 5 female, aged from 14 to 83 years. Within the 12 cases, there were 4 cases with pancreatic carcinoma, 5 cases with pancreatic solid pseudopapillary tumor, 2 cases with pancreatic serous cystadenoma, 1 case with pancreatic cyst (ductal epithelial papillary hyperplasia). RESULTS: Nine tumors which had been regarded as removable pre-operatively with MI-3DVS were removed successfully. Three patients who were considered unresectable by other hospitals with CT were operated successfully with MI-3DVS. The other 3 patients' tumors were actually not able to be removed as pre-operative evaluation. CONCLUSION: MI-3DVS plays an important role in diagnosis and assessment of resectability of pancreatic tumor.

Analysis of age-related changes in midfacial fat compartments in Asian women using computed tomography.

BACKGROUND: Volume restoration is no more a fresh theory for midfacial rejuvenation. However, lack of knowledge regarding the natural ageing process of fat compartments often leads to an insufficient or excessive clinical result. The aim of this study is to reveal the age-related changes in midfacial fat compartments and the correlation between midfacial grooves and the related fat compartments. METHODS: This study included 60 Asian females in defined age-based categories. The thickness of the infraorbital fat compartment, the nasolabial fat compartment, and the cheek fat compartments were measured using computed tomography (CT) images. Analysis of correlations between midfacial grooves and the related fat compartments was performed using the SPSS software. RESULTS: A tendency of thickening in the infraorbital fat and nasolabial fat compartments with age was observed. The superficial layer of cheek fat compartments was found to be thinner, and a similar tendency was observed in the medial part of deep medial cheek fat. However, it was thicker in the lateral part of deep medial cheek fat. There was a negative correlation between the fat thickness of deep medial cheek fat and both the severity of tear trough deformity and the nasolabial fold. A positive correlation between the lower third of the nasolabial fat compartment and the severity of the nasolabial fold was found as well. CONCLUSION: Different midfacial fat compartments tended to undergo selective hypertrophy or atrophy with ageing. The findings of this study suggested that augmentation of the deflated fat compartment and liposuction of the hypertrophic fat compartment can provide a more natural effect in facial rejuvenation.

Evaluation of three hormonal protocols for anovulatory lactating cows under regulations restricting the use of estrogenic compounds.

When European Union regulations restricted the use of estrogenic compounds in food-producing animals, refined hormonal protocols were no longer applicable for anovulatory cows. However, Ovsynch and its adaptations are routinely and uniformly applied to all cows regardless of ovarian function. To evaluate their efficacy on anovulatory cows, 143, 147 and 144 anovulatory cows received Ovsynch, Presynch and G6G protocols, respectively. In comparison, 150 cyclic cows were bred without using a synchronized protocol. Results showed that cows in the Presynch group had luteolysis responding to the last prostaglandin F2α (PGF2α ) injection greater than the Ovsynch group. The serous progesterone levels at the first gonadotropin-releasing hormone of Ovsych and the last PGF2α injection was greater in the G6G group than the other two hormonal treatment groups. Concentrations of Ca2+ and total protein in cervical mucus in all three hormone-treated groups before artificial insemination (AI) were significantly different from the controls. The G6G group obtained a greater pregnancy rate compared with Ovsynch and Presynch, but significantly less than the controls. For open cows in the Ovsynch group, estrus rate within 24 days after the first AI was significantly less than the controls. In conclusion, the G6G treatment resulted to better reproductive performance in anovulatory cows.

Risk factors analysis of mirror aneurysms: A multi-center retrospective study based on clinical and demographic profile of patients.

As a special subgroup of multiple intracranial aneurysms, mirror aneurysms are located bilaterally on the corresponding intracranial arteries. The current study sought to compare the clinical and demographic features of patients harboring mirror aneurysm, and to elucidate the corresponding risk factors. We performed a retrospective cohort study of 2641 intracranial aneurysms patients, who were admitted to our hospitals between January 2005 and June 2014. Patients were subdivided into three groups based on the inclusion criteria: (i) single (n=2250); (ii) non-mirror multiple (n=285); and (iii) mirror aneurysms (n=106). Clinical and demographic files of the three groups were collected and compared, and medical histories including stroke, hyperlipemia, hypertension, hyperglycemia, valvular heart disease were considered as potential risk factors. Potential morphological reasons for mirror cerebral aneurysms rupture, including aneurysms size, irregular walls and cerebral hemispheric dominance, were also compared. Our data showed that the male to female ratio of mirror aneurysms patients was 1:3.61, which was significantly different from that of single aneurysm (1:1.27) and multiple aneurysms (1:2.00). The prevalence of mirror aneurysms in women is higher than that in men (P<0.001). Older patients (especially 60-69 years old) also appear to be more vulnerable to mirror aneurysm than single aneurysm (P<0.001). In 84 mirror aneurysm patients the aneurysms were located on the internal carotid arteries (79.2%), most typically at the PComA or in the Cavernous ICA. Patients with medical history of hyperlipemia appear to have an increased risk of harboring mirror aneurysms. Larger aneurysm size and presence of an irregular aneurysm wall appear to be the morphological factors that predispose for mirror aneurysms rupture.

BIM EL-mediated apoptosis in cumulus cells contributes to degenerative changes in aged porcine oocytes via a paracrine action.

Whether cumulus cells (CCs) contribute to oocyte aging remains controversial; in that regard, little is known about biochemical processes of gene expression in CCs surrounding aged oocytes. The objective was to elucidate contributions of CCs to porcine oocyte aging and degeneration, apoptosis and BIM expression in CCs during oocyte aging in vitro. When culture of cumulus oocyte complexes (COCs) was prolonged (68 h, which resulted in 24 h of aging), the rate of blastocyst formation following electro-activation was lower than that of oocytes aged without CCs (2.6 ± 0.1 vs 13.5 ± 1.3%, mean ± SEM; P < 0.05). In addition, the presence of CCs significantly accelerated spontaneous fragmentation of oocytes following prolonged (92 h) culture. Apoptotic CCs were present in COCs cultured for 68 h, and the abundance of Bim mRNA in CCs progressively increased after 56 h of culture (P < 0.05). Based on immunofluorescence, BIM protein expression was up-regulated in CCs surrounding aged oocytes; furthermore, quantification (Western blot) of BIM(EL) protein progressively increased after 56 h of culture. Lastly, in a series of experiments to elucidate the signal pathway, blocking gap junctions (with 1-octanol) during aging did not eliminate the effect of CCs on accelerating oocyte aging, but prolonged co-culture of denuded oocytes with COCs after in vitro maturation reduced blastocyst rate relative to culture of denuded oocytes aged alone (4.15 ± 0.1 vs 11.0 ± 0.7%, P < 0.05). We concluded that apoptotic CCs, in which BIM(EL) up-regulation was involved, accelerated oocyte aging and degeneration in vitro via a paracrine action.

Acetylation of H4K12 in porcine oocytes during in vitro aging: potential role of ooplasmic reactive oxygen species.

Deterioration in the quality of mammalian mature oocytes during metaphase-II (M-II) arrest is called "oocyte aging". Although histone acetylation may affect the progression of aging in murine oocytes, the mechanism is unknown. The objective was to determine the role of ooplasmic reactive oxygen species (ROS) in acetylation of histone H4 at lysine 12 (acH4K12) in porcine aged oocytes in vitro. Based on immunostaining with a specific antibody, acetylation of H4K12 in porcine oocytes increased during in vitro aging, which coincided with changing patterns of ooplasmic ROS content. Furthermore, both hydrogen peroxide (H(2)O(2)), and the mitochondrial membrane potential disrupter, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which can moderately elevate oocyte ROS content, significantly increased acetylation levels of H4K12 in porcine oocytes. It was noteworthy that acetylation in the CCCP group was decreased when ROS was counteracted by cysteine, a common antioxidant. In addition, the intracellular mRNA abundance of acetyltransferase gene HAT1 in aged and H(2)O(2) treated oocytes was higher than in M-II phase oocytes, suggesting that HAT1 was involved in this reaction. After parthenogenetic activation, a lower proportion of oocytes developed to the blastocyst stage after CCCP or H(2)O(2) treatment when compared with M-II phase oocytes (20 and 0% for CCCP and H(2)O(2) groups, respectively, versus 42% for the M-II group, P < 0.05). In conclusion, elevated levels of H4K12 acetylation were attributed to increased ooplasmic ROS content during porcine oocyte aging in vitro.

Synthesis of novel sulfanilamide-derived 1,2,3-triazoles and their evaluation for antibacterial and antifungal activities.

A series of novel sulfanilamide-derived 1,2,3-triazole compounds were synthesized in excellent yields via 1,3-dipolar cycloaddition and confirmed by MS, IR and NMR spectra as well as elemental analyses. All the compounds were screened in vitro for their antibacterial and antifungal activities. Preliminary results indicated that some target compounds exhibited promising antibacterial potency. Especially, 4-amino-N-((1-dodecyl-1H-1,2,3-triazol-4-yl)methyl) benzenesulfonamide, N-((1-(2,4-dichlorobenzyl)- 1H-1,2,3-triazol-4-yl)methyl)-4-aminobenzenesulfonamide and 4-amino-N-((1-(2,4-difluorobenzyl)- 1H-1,2,3-triazol-4-yl)methyl) benzenesulfonamide were found to be the most potent compounds against all the tested strains except for Candida albicans (ATCC76615) and Candida mycoderma.