Prognostic significance of bone marrow 2-[18F]-fluoro-2-deoxy-d-glucose uptake in diffuse large B-cell lymphoma: relation to iliac crest biopsy results.

AIM: To investigate the prognostic significance of bone marrow (BM) 2-[18F]-fluoro-2-deoxy-d-glucose (FDG) uptake in relation to posterior iliac crest BM biopsy (BMB) results in diffuse large B-cell lymphoma (DLBCL). MATERIALS AND METHODS: Pretreatment integrated positron-emission tomography(PET)/computed tomography (CT) images of 512 DLBCL patients who underwent BMB and received rituximab combined with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) chemotherapy were analysed retrospectively. BM uptake was assessed visually and by maximum standard uptake value (SUVmax). Associations with lymphoma-specific survival (LSS) were assessed using Kaplan-Meier and Cox regression analyses. RESULTS: FDG(+) BM was observed in 64 cases (41 focal, 12 heterogeneous, 11 diffuse). This finding distinguished iliac crest involvement (positive in 59 and negative in 453) with 89.6% accuracy (459/512) and 93.6% specificity (424/453). In BMB(+) patients, BM-to-liver SUVmax ratio >1.8 concurred perfectly with FDG(+) BM. During 52 months of follow-up, there were 156 lymphoma-related deaths. In the entire population, multivariate analysis revealed high International Prognostic Index (IPI; p<0.001), old age (p=0.003), bulky disease (p=0.011), BMB(+) (p=0.028), and FDG(+) BM (p=0.019) as independent predictors of worse LSS. In the BMB(+) subgroup, high National Comprehensive Cancer Network-revised IPI (NCCN-IPI; p=0.029) and FDG(+) BM (p=0.008) were significant independent predictors. Among BMB(+) patients with low to low-intermediate NCCN-IPI, FDG(+) BM was associated with significantly worse 2-year LSS (33.3% versus 100%; p=0.017). The same was true among those with high-intermediate NCCN-IPI (34.7% versus 76.9%.; p=0.026). CONCLUSION: Increased BM FDG in DLBCL is a predictor of worse LSS independent of BMB results and other prognostic variables including IPI/NCCN-IPI.

Pre-treatment 18F-FDG PET-based radiomics predict survival in resected non-small cell lung cancer.

AIM: To assess the prognostic value of 2-[18F]-fluoro-2-deoxy-d-glucose (FDG) positron-emission tomography (PET)-based radiomics using a machine learning approach in patients with non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Ninety-three patients with stage I-III NSCLC who underwent combined PET/computed tomography (CT) followed by curative resection. A total of 35 unique quantitative radiomic features was extracted from the PET images, which included imaging phenotypes such as pixel intensity, shape, and texture. Radiomic features were ranked based on score according to their correlation with disease recurrence status within a 3-year follow-up. The recurrence risk classification performances of machine learning algorithms (random forest, neural network, naive Bayes, logistic regression, and support vector machine) using the 20 best-ranked features were compared using the areas under the receiver operating characteristic curve (AUC) and validated by the random sampling method. RESULTS: Contrast and busyness texture features from neighbourhood grey-level difference matrix were found to be the two best predictors of disease recurrence. The random forest model obtained the best performance (AUC: 0.956, accuracy: 0.901, F1 score: 0.872, precision: 0.905, recall: 0.842), followed by the neural network model (AUC: 0.871, accuracy: 0.780, F1 score: 0.708, precision: 0.755, recall: 0.666). CONCLUSION: A PET-based radiomic model was developed and validated for risk classification in NSCLC. The machine learning approach with random forest classifier exhibited good performance in predicting the recurrence risk. Radiomic features may help clinicians to improve the risk stratification for clinical practice.

Evaluation of a diagnostic 18F-FDG PET/CT strategy for differentiating benign from malignant retroperitoneal soft-tissue masses.

AIM: To investigate the optimal combined 2-[18F]-fluoro-2-deoxy-d-glucose (FDG) positron-emission tomography (PET)/computed tomography (CT) diagnostic criteria for distinguishing between benign and malignant retroperitoneal soft-tissue masses (RPMs). MATERIALS AND METHODS: A total of 74 patients (M:F=34:40; age, 53±13.2 years) who underwent FDG PET/CT for the initial work-up of RPMs were included. The maximum standardised uptake value (SUVmax), tumour size, presence of fat or calcifications and separated hypermetabolic lesions were included as PET/CT diagnostic parameters. Receiver-operating characteristic (ROC) curves were used to compare the diagnostic performance. RESULTS: The final pathological diagnoses included 52 malignant and 22 benign tumours. High SUVmax (>4.8) and large size (>13 cm) favoured malignancy, and yielded a diagnostic accuracy and AUC of 64.9%, 0.820±0.059, and 68.9%, 0.738±0.061, respectively. In a subgroup of RPMs with a fat component, both SUVmax and size were significantly different between benign and malignant RPM, which yielded a diagnostic accuracy and AUC of 91%, 0.977±0.024 (cut-off, 1.9 cm) and 87.9%, 0.865±0.072 (cut-off, 13 cm), respectively. In a subgroup without a fat component, only SUVmax was significantly different with an accuracy of 90.2% and AUC of 0.919±0.043. The optimal diagnostic flow by combining SUVmax and tumour size after dividing patients into two groups according to the presence of fat showed a sensitivity of 90.4%, a specificity of 95.5%, and an accuracy of 91.9%. CONCLUSIONS: The combination of SUVmax and size according to the presence of a fat component may be the optimal PET/CT diagnostic criteria for distinguishing benign and malignant RPMs.

Intratumoural heterogeneity measured using FDG PET and MRI is associated with tumour-stroma ratio and clinical outcome in head and neck squamous cell carcinoma.

AIM: To evaluate the association between the tumour-stroma ratio and intratumoural heterogeneity measured using 2-[18F]-fluoro-2-deoxy-d-glucose (FDG) positron-emission tomography (PET) and magnetic resonance imaging (MRI), and further investigate the prognostic significance of imaging biomarkers in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: Textural-based imaging parameters of the primary tumour were extracted in 44 patients. In addition, the difference between the minimum and maximum apparent diffusion coefficient (ADC) values (ADCdiff) was calculated on MRI. The relationships between the tumour-stroma ratio and imaging parameters were evaluated. The associations between imaging parameters and recurrence-free survival (RFS) were assessed using Cox proportional hazard regression models. RESULTS: Coarseness (r=-0.382) on PET and ADCdiff (r=0.534) on MRI were significantly correlated with the proportion of stroma. The best imaging biomarkers for the 2-year RFS prediction were coarseness (AUC=0.741) and ADCdiff (AUC=0.779). Multivariate analysis showed that coarseness (hazard ratio=10.549, 95% confidence interval=2.544-43.748, p=0.001) was an independent prognostic factor for RFS. CONCLUSION: Heterogeneity imaging parameters are significantly associated with the tumour-stroma ratio. These imaging biomarkers may help to facilitate the risk stratification for tumour recurrence in HNSCC.

Effect of rapamycin treatment during post-activation and/or in vitro culture on embryonic development after parthenogenesis and in vitro fertilization in pigs.

This study investigated the effects of early induction of autophagy on embryonic development in pigs. For this, oocytes or embryos were treated with an autophagy inducer, rapamycin (RP), during post-activation (Pa), in vitro fertilization (IVF) and/or in vitro culture (IVC). When parthenogenesis (PA) embryos were untreated (control) or treated with various concentrations of RP for 4 hr during Pa, 100 nm RP showed a higher blastocyst formation (48.8 ± 2.7%) than the control (34.6 ± 3.0%). When PA embryos were treated during the first 24 hr of IVC, blastocyst formation was increased (p < .05) by 1 and 10 nm RP (61.9 ± 3.0 and 59.6 ± 3.0%, respectively) compared to the control (43.2 ± 1.8%) and 100 nm RP (47.8 ± 3.2%), with a higher embryo cleavage in response to 10 nm RP (87.3 ± 2.4%) than the control (74.1 ± 3.2%). RP treatment during IVC and Pa + IVC showed increased blastocyst formation (44.7 ± 2.5 and 44.1 ± 2.0%, respectively) compared to the control (33.2 ± 2.0%). In addition, RP treatment during Pa and/or IVC increased glutathione content and inversely reduced reactive oxygen species. In IVF, RP treatment for 6 hr during IVF significantly increased embryonic development (34.0 ± 2.6%) compared to the control (24.8 ± 1.6%), but treatment during IVC for 24 hr with RP did not (23.0 ± 3.8%). Autophagy was significantly increased in PA oocytes by the RP treatment during Pa but not altered by the treatment during the first 24 hr of IVC. Overall, RP treatment positively regulated the pre-implantation development of pig embryos, probably by regulating cellular redox state and stimulating autophagy.

Cilostazol Improves Developmental Competence of Pig Oocytes by Increasing Intraoocyte Cyclic Adenosine Monophosphate Level and Delaying Meiotic Resumption.

Cilostazol (CLZ) is a cyclic adenosine monophosphate (cAMP) modulator that influences the steady state of the meiotic stage. This study was conducted to determine the effects of CLZ treatment during in vitro maturation (IVM) on developmental competence of pig oocytes. Immature oocytes were exposed to 0 (control), 0.5, 2 and 4 µm CLZ during the first 22 h of IVM. Nuclear maturation, intraoocyte glutathione content and embryo cleavage after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were not influenced by CLZ at any concentrations. However, 4 µm CLZ significantly (p < 0.05) improved blastocyst formation after PA (52.1% vs 38.7-46.0%) and SCNT relative to other concentrations (40.8% vs 25.0-30.7%). The mean cell numbers of SCNT blastocysts were significantly increased by 4 µm CLZ compared to the control (42.6 cells vs 35.3 cells/blastocyst). CLZ treatment significantly increased the intraoocyte cAMP level and effectively arrested oocytes at the germinal vesicle (GV) and GV break down stages compared to the control (74.5% vs 45.4%). Our results demonstrated that improved developmental competence of PA and SCNT pig embryos occurred via better synchronization of nuclear and cytoplasmic maturation induced by increased cAMP and delayed meiotic resumption after CLZ treatment.

Interleukin-18, transforming growth factor-ß, and vascular endothelial growth factor gene polymorphisms and susceptibility to primary glomerulonephritis.

Several studies have showed an association of gene polymorphisms with the development of glomerulonephritis (GN). We investigated the effects of gene polymorphisms on the development of GN by analyzing polymorphisms in the interleukin (IL)-18, transforming growth factor (TGF)-ß, and vascular endothelial growth factor (VEGF) genes in Korean patients with primary GN. The study included 146 normal subjects (controls) and 100 patients diagnosed with primary GN by kidney biopsy. The gene polymorphisms A-607C and G-137C in IL-18, C-509T and T869C in TGF-ß1, and C-2578A and C405G in VEGF were investigated in DNA extracted from peripheral blood. Significant differences were observed between the GN and control groups in the genotype and allele frequencies of A-607C IL-18 and C405G VEGF. The frequencies of the IL-18-607CC genotype [P = 0.001, odds ratio (OR) = 2.473] and the VEGF 405GG genotype (P = 0.001, OR = 2.473) were significantly increased in the GN group. The combination of IL-18-607CC+ and VEGF 405GG+ genotypes had a higher risk for developing GN in comparison with the combination of IL-18-607CC- and VEGF 405GG- genotypes (P < 0.001, OR = 8.642). In the haplotype analysis of the IL-18 gene, the CG haplotype was significantly more frequent in the GN group than the control group (61.5% vs 46.9%, P = 0.002). These results show that the -607CC genotype of the IL-18 gene and the 405GG genotype of the VEGF gene are associated with susceptibility to and the development of primary GN.

Establishment and characterization of embryonic stem-like cells from porcine somatic cell nuclear transfer blastocysts.

This study was aimed to establish embryonic stem (ES)-like cells from blastocysts derived from somatic cell nuclear transfer (SCNT) in pig. Somatic cells isolated from both day-30 fetus and neonatal cloned piglet were used for donor cells. A total of 60 blastocysts (46 and 14 derived from fetal and neonatal fibroblast donor cells, respectively) were seeded onto a mitotically inactive mouse embryonic fibroblast (MEF) monolayer and two ES-like cell lines, one from each donor cell type, were established. They remained undifferentiated over more than 52 (fetal fibroblast-derived) and 48 (neonatal fibroblast-derived) passages, while retaining alkaline phosphatase activity and reactivity with ES specific markers Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-4, TRA-1-60 and TRA-1-81. These ES-like cells maintained normal diploid karyotype throughout subculture and successfully differentiated into embryoid bodies that expressed three germ layer-specific genes (ectoderm: beta-III tubulin; endoderm: amylase; and mesoderm: enolase) after culture in leukemia inhibitory factor-free medium. Microsatellite analysis confirmed that they were genetically identical to its donor cells. Combined with gene targeting, our results may contribute to developing an efficient method for producing transgenic pigs for various purposes.

Characterization of congenital lymphatic and blood vascular malformations in the head and neck using blood pool scintigraphy and spect.

The purpose of this study was to investigate the usefulness and diagnostic efficacy of blood pool (BP) scintigraphy and SPECT for characterizing congenital vascular malformations (CVMs) in the head and neck area. A total of 154 patients suspected of having head and neck CVMs underwent whole-body BP scintigraphy and head and neck BP SPECT using 99mTc-labeled red blood cells. Based on SPECT findings, CVMs were classified into lymphatic malformation/ non-(blood) vascular disease (LM/NVD, no distinct uptake), arterio-venous malformation (AVM, abnormal uptake in lesions and asymmetrically increased jugular vein uptake on ipsilateral side), venous malformation (VM, strong uptake in lesions with symmetric jugular vein uptake), and veno-lymphatic malformation (VLM, no or mild uptake on lesions with symmetric jugular vein uptake). The sensitivities and specificities of BP SPECT for diagnosing each subtype of head and neck CVM were 100% (13/13) and 97.1% (137/141) for LM/NVD, 61.1% (22/36) and 99.1% (117/118)for AVM, 91.7% (88/96) and 79.3% (46/58) for VM, and 55.6% (5/9) and 93.7% (136/145) in VLM, respectively. The overall accuracy for characterizing CVMs by head and neck BP SPECT was 83.1% (128/154). In conclusion, BP SPECT is a useful method for classifying CVMs in the head and neck area due to its high diagnostic efficacy.

Demetylation of the sex-determining region Y gene promoter and incidence of disorder of sex development in cloned dog males.

Canine cloning is occasionally accompanied by abnormal sexual development. Some male donor cells produce cloned pups with female external genitalia and complete male gonadal dysgenesis, which is classified as an XY disorder of sex development (XY DSD). In this study, we examine the potential of 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, to reduce the phenotypic abnormality XY DSD in somatic cell nuclear transfer (SCNT)- derived pups. We used a 9-year-old normal male German Shepherd dog as a cell donor. Donor cells were treated with 10 nM 5-aza-dC for 4 days before being used for SCNT. At the same stage of cell development, significantly lower levels of DNA methylation of the sex-determining region Y (SRY) promoter was observed in the treated donor cells compared to that in the untreated cells (95.2% versus 53.3% on day 4 for the control and treated groups, respectively). No significant differences were observed in the control or treatment groups concerning fusion rate, pregnancy rate (30 days or entire period), the number of pups, or the incidence of XY DSD. However, more XY DSD dogs were observed in the control group (31.25%) than in the treatment group (14.29%). Hypermethylation of the SRY promoter was observed in the XY DSD cloned pups in both the treatment (84.8%) and control groups (91.1 ± 1.4%) compared to the methylation level in the phenotypically normal male pups of the treatment (23.2 ± 20.9%) and control groups (39.1 ± 20.1%). These results suggest that 5-aza-dC treatment of donor cells can reduce the methylation level of the SRY promoter in donor cells, and thus, 5-aza-dC is advantageous for reducing the incidence of XY DSD in canine cloning.

Uterine expression of epidermal growth factor family during the course of pregnancy in pigs.

To stably maintain pregnancy, several genes are expressed in the uterus. In particular, the endometrial expression of genes encoding growth factors appears to play a key role in maternal-foetal communication. The previous studies characterized the endometrial expression kinetics of the genes encoding epidermal growth factor (EGF), its receptor (EGFR), transforming growth factor-alpha (TGF-alpha), amphiregulin (Areg), heparin-binding (Hb) EGF and calbindin-D9k (CaBP-9k) in pigs during implantation. Here, we further characterized the expression patterns of these molecules during the entire porcine pregnancy. Porcine uteri were collected at pregnancy days (PD) 12, 15, 30, 60, 90 and 110 and subjected to RT-PCR. EGF and EGFR showed similar expression patterns, being highly expressed around implantation and then disappearing. TGF-alpha and Areg expression levels rose steadily until they peaked at PD30, after which they gradually decreased to PD12 levels. This Areg mRNA expression pattern was confirmed by real-time PCR and similar Areg protein expression patterns were observed. Immunohistochemical analysis of PD60 uteri revealed Areg in the glandular and luminal epithelial cells. Hb EGF was steadily expressed throughout the entire pregnancy, while CaBP-9k was expressed strongly on PD12, and then declined sharply on PD15 before recovering slightly for the remainder of the pregnancy. Thus, the EGF family may play a key role during implantation in pigs. In addition, CaBP-9k may help to maintain uterine quiescence during pregnancy by sequestering cytoplasmic Ca(2+).

Characterization of congenital vascular malformation in the extremities using whole body blood pool scintigraphy and lymphscintigraphy.

The purpose of this study was to investigate the clinical usefulness of combined whole body blood pool scintigraphy (WBBPS) and lymphscintigraphy (LS) in the characterization of patients with congenital vascular malformations (CVMs) of the extremities. Subjects included 134 patients who underwent Tc-99m RBC WBBPS and Tc-99m filtered tin colloid (or antimony sulfur colloid) LS on initial diagnosis. Scintigraphic results were interpreted as arteriovenous malformations (AVMs), venolymphatic malformations (VLMs), lymphatic malformations (LMs), and venous malformations (VMs). Final diagnosis of the type of vascular malformation was determined by physical examination, magnetic resonance imaging (MRI), angiography, duplex ultrasonography, and/or biopsy results. The final diagnosis demonstrated that 14 of the study subjects had an AVM, 29 had a HLM, 20 had a LM, and 71 had a VM. The sensitivity of WBBPS and LS in the characterization of CVM was 85.7% (12/14) for AVMs, 96.6% (28/29) for VLMs, 95.0% (19/20) for LMs, and 88.7% (63/71) for VMs. The specificity was 100% for AVMs (120/120), 91.4% for VLMs (96/105), 99.1% for LMs (113/114), and 98.4% for VMs (62/63). The overall accuracy of WBBPS and LS was 91.0% (122/134). Our results show that combination of WBBPS with LS can characterize extremity CVMs in patients with high diagnostic accuracy, and may thus be useful for making optimal treatment decisions.

Lymphscintigraphy predicts response to complex physical therapy in patients with early stage extremity lymphedema.

We investigated whether baseline lymphscintigraphic findings can predict long-term response to complex physical therapy (CPT) in patients with early stage extremity lymphedema. Twenty patients with unilateral extremity lymphedema of clinical stage I or II underwent CPT after baseline lymphscintigraphy. Therapeutic responses (good vs. poor) were evaluated at 1 year post-CPT based on changes in skin status and subjective symptoms, and percent volume reductions and compared with clinical factors and lymphscintigraphic findings. Eleven patients showed good response to CPT with significant volume reduction of edematous extremities, and no significant volume reduction was observed in the remaining 9. Patients with good or poor responses to CPT showed no significant differences in terms of clinical variables. However, significant differences were observed between the lymphscintigraphic findings of these patients. More specifically, a lymphscintigraphic finding of main lymphatic vessels without collateral lymphatic vessels was the best predictor for a good response to CPT; the sensitivity, specificity and accuracy of this lymphscintigraphic finding is 91% (10/11), 100% (9/9) and 95% (19/20), respectively. In patients with unilateral extremity lymphedema of early stage, baseline lymphscintigraphy may usefully predict long-term response to CPT.

A micromachined electrochemical sensor for free chlorine monitoring in drinking water.

In this work, we designed, fabricated and tested a disposable, flow-through amperometric sensor for free chlorine determination in water. The sensor is based on the principle of an electrochemical cell. The substrate, as well as the top microfluidic layer, is made up of a polymer material. The advantages include; (a) disposability from low cost; (b) stable operation range from three-electrode design; (c) fluidic interconnections that provide on line testing capabilities; and (d) transparent substrate which provides for future integration of on-chip optics. The sensor showed a good response and linearity in the chlorine concentration ranging from 0.3 to 1.6 ppm, which applies to common chlorination process for drinking water purification.

Alpha-linolenic acid treatment during oocyte maturation enhances embryonic development by influencing mitogen-activated protein kinase activity and intraoocyte glutathione content in pigs.

The purpose of this study was to examine the effects of alpha-linolenic acid (ALA) treatment during in vitro maturation (IVM) on nuclear maturation, intraoocyte glutathione (GSH) content, meiotic progression, and developmental competence after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. Medium-199 containing 10% (vol/vol) porcine follicular fluid (PFF; PPF control) or 0.4% (wt/vol) fatty acid-free BSA (BSA control) was used for IVM. The proportion of oocytes reaching the metaphase II (MII) stage was not influenced by ALA treatment at various concentrations (50, 100, and 200 µ). However, treatment with 100 µ ALA significantly increased ( < 0.05) intraoocyte GSH content (1.19 vs. 1.00 and 0.92 pixels per oocyte, comparing the treated oocytes, BSA control, and PFF control, respectively) and embryonic development to the blastocyst stage after PA (47.1 vs. 35.5 and 35.2%) and SCNT (31.4 vs. 23.9 and 24.3%). ALA treatment (100 µ) accelerated oocyte maturation, and a higher proportion of ALA-treated oocytes (89.6%) reached the MII stage than did the untreated controls (75.5%) at 33 h of IVM. Mitogen-activated protein kinase kinase inhibitor (U0126) treatment during IVM inhibited nuclear maturation and embryonic development after PA. However, 100 µ ALA completely counteracted the suppressive effect of U0126 on nuclear maturation and partially counteracted the effect on blastocyst formation. Our results demonstrate that treatment with 100 µ ALA during IVM improves developmental competence by accelerating nuclear maturation and also influencing cytoplasmic maturation, such as increased GSH content in IVM oocytes.

Prognostic relevance of pretransplant Deauville score on PET-CT and presence of EBV DNA in patients who underwent autologous stem cell transplantation for ENKTL.

High-dose chemotherapy and autologous stem cell transplantation (ASCT) for extranodal natural killer/T-cell lymphoma (ENKTL) is a reasonable option for a subset of patients. The impact of response status, according to positron emission tomography/computed tomography (PET/CT) results and/or presence of circulating EBV DNA prior to ASCT, has not yet been established. We analyzed 27 ENKTL patients with pre-ASCT circulating EBV DNA who had undergone pre-ASCT PET/CT between 2009 and 2014. We classified patients into two groups based on the result of pretransplantation assessment: a favorable risk group (pretransplant five-point Deauville score (DS) of 1-2 based on PET/CT and no detectable EBV DNA) and an unfavorable risk group (DS 1-2 with detectable EBV DNA, DS 3-5 with or without detectable EBV DNA). After a median follow-up of 37 months, overall survival and PFS were significantly different between the two groups (median OS: not reached for favorable risk group vs 7.0 months for unfavorable risk group, P=0.017; median PFS: 16.0 vs 5.0 months, P=0.019). Multivariate analysis revealed that pre-ASCT DS and EBV DNA was the only independent prognostic factor considering stage, IPI and NKPI. Precise assessment of the status of disease before transplantation may provide more benefit from ASCT to ENKTL patients.

Effect of maturation media and oocytes derived from sows or gilts on the development of cloned pig embryos.

In order to develop a culture system and recipient cytoplasm that could improve the developmental competence of somatic cell nuclear transfer (SCNT) embryos for successful cloning of pigs, we evaluated the effect of donor oocytes and in vitro maturation (IVM) media on maturation of oocytes and developmental competence of SCNT embryos. In Experiment 1, oocytes derived from sows or gilts were matured in two IVM media (TCM-199 versus NCSU-23) and maturation of oocytes was evaluated by the status of chromatin configuration, the diameter of matured oocytes, the thickness of the zona pellucida, and the size of the perivitelline space (PVS). Sow oocytes matured in TCM-199 (S-TCM group) and NCSU-23 (S-NCSU group) showed significantly higher (P<0.05) maturation rates (S-TCM and S-NSCU, 86+/-4 and 82+/-4%, respectively) when evaluated by metaphase-II status than the gilt oocytes matured in TCM-199 (G-TCM group, 71+/-3%) and in NCSU-23 (G-NCSU-23 group, 71+/-3%). Oocyte diameter, the thickness of the zona pellucida, and the perivitelline space of sow oocytes (S-TCM and S-NCSU) were larger than those of gilt oocytes (G-TCM and G-NCSU) after IVM (P<0.05). In Experiment 2, SCNT was performed, using in vitro-matured oocytes from each group as recipient cytoplasm and porcine fetal fibroblasts as karyoplasts. The reconstructed embryos were electrically fused and activated, and cleavage and blastocyst formation were monitored under a stereomicroscope. The total cell number of flattened blastocysts stained with 5 microM bisbenzimide on day 7 were counted. In addition, in vitro matured non-enucleated oocytes were also electrically activated (parthenogenetic activation) and pronuclear formation was monitored. No difference in pronuclear formation rate after parthenogenetic activation and fusion rate after SCNT was observed among experimental groups. A significantly higher cleavage rate (P<0.05) was observed in S-TCM (69+/-4%) when compared with only G-NCSU (58+/-4%), but not with G-TCM (60+/-4%) or S-NCSU (68+/-4%). The rate of blastocyst formation was significantly higher (P<0.05) in sow oocytes (24% in S-TCM and S-NCSU), when compared to that observed in G-TCM (15%), and G-NCSU (14%). When the same source of oocytes was used, there was no significant difference in rate of blastocyst formation in the two culture media. Total cell number of blastocysts were not significantly different among experimental groups. In conclusion, the present study clearly demonstrated that sow oocytes have a greater developmental competence than gilt oocytes, regardless of the maturation medium examined.

Effect of particle size and sodium ion concentration on anaerobic thermophilic food waste digestion.

Serial basic tests were conducted for the determination of fundamental kinetics and for the actual application of kinetic parameters to food waste digestion with precise measurement of methane production. The effects of food particle size and sodium ion concentration on the anaerobic thermophilic food waste digestion process were investigated. Results of serial tests for the determination of fundamental kinetic coefficients showed the value of k (maximum substrate utilization rate coefficient) and KS (half-saturation coefficient) as 0.24 hr-1 and 700 mg/L, respectively, for non-inhibiting organic loading range. The substrate inhibition coefficient (Ki) was 1000 mg/L for inhibiting organic loading range. No inhibition effect was shown until 5 g/L of sodium ion concentration was applied to the serum bottle reactor. However, the volume of methane gas was decreased gradually to the concentrations of more than 5 g/L of sodium ion applied. All sizes of food waste particle had the same constants (A: 0.45) but the maximum substrate utilization rate constant (kHA) was inversely proportional to particle size. As average particle size increased from 1.02 mm to 2.14 mm, kHA decreased from 0.0033 hr-1 to 0.0015 hr-1. Result reveals that particle size is one of the most important factors in anaerobic food waste digestion.

Effects of porcine granulocyte-macrophage colony-stimulating factor on porcine in vitro-fertilized embryos.

This study investigated the effects of porcine granulocyte-macrophage colony-stimulating factor (pGM-CSF) on the developmental potential of porcine in vitro-fertilized (IVF) embryos in chemically and semidefined (with BSA) medium. In experiment 1, zygotes were treated with different concentrations of pGM-CSF (0, 2, 10, 100 ng/mL). The results indicated that 10 ng/mL pGM-CSF significantly (P < 0.05) increased blastocyst development and total cell number (15.1% and 53.5, respectively) compared with the control (6.1%, and 38.8, respectively). Comparing blastocyst formation, early and expanded blastocyst formation was significantly higher in the 10 ng/mL-pGM-CSF group than in the control on Days 6 and 7 of the culture period. However, there was no significant difference in cleavage rate. Experiment 2 demonstrated that pGM-CSF influenced the percentage of blastocyst formation and total cell number when pGM-CSF was added during Days 4 to 7 (14.6% and 53.9, respectively) or Days 0 to 7 (15.2% and 54.0, respectively) compared with the control (7.8% and 43.1, respectively) and compared with Days 0 to 3 (8.7% and 42.5, respectively). Similarly, early blastocyst formation rates were significantly higher at Days 4 to 7 than in the control, and expanded blastocyst formation was significantly higher at Days 4 to 7 or Days 0 to 7. No significant difference in cleavage rates appeared among the groups. In experiment 3, in the presence of BSA, pGM-CSF also increased the percentage of embryos that developed to the blastocyst stage and the total cell number (20.3% and 59.8, respectively) compared with the control (14.9% and 51.4, respectively), whereas there was no significant difference in cleavage rate. Experiment 4 found that the total cell number and the number of cells in the inner cell mass (ICM) were significantly increased compared with the control when zygotes were cultured in either porcine zygotic medium (PZM)-3 or PZM-4 supplemented with 10 ng/mL pGM-CSF. The number of trophectoderm (TE) cells was significantly higher in PZM-3 medium supplemented with pGM-CSF than in the control, and the number tended to increase (P = 0.058) in PZM-4 medium supplemented with pGM-CSF. The ratio of inner cell mass to trophectoderm cells was significantly higher in PZM-4 supplemented with 10 ng/mL pGM-CSF, but not in PZM-3. In experiment 5, it was found that the male pronuclear formation rate, monospermic penetration and sperm/oocyte were 95.4%, 37.2%, and 2.4, respectively. Together, these results suggest that pGM-CSF may have a physiological role in promoting the development of porcine preimplantation embryos and regulating cell viability and that addition of pGM-CSF to IVC medium at Days 4 to 7 or 0 to 7 improves the developmental potential of porcine IVF embryos.

Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.

As stem cells are capable of self-renewal and can generate differentiated progenies for organ development, they are considered as potential source for regenerative medicine and tissue replacement after injury or disease. Along with this capacity, stem cells have the therapeutic potential for treating human diseases including cancers. According to the origins, stem cells are broadly classified into two types: embryonic stem cells (ESCs) and adult stem cells. In terms of differentiation potential, ESCs are pluripotent and adult stem cells are multipotent. Amnion, which is a membranous sac that contains the fetus and amniotic fluid and functions in protecting the developing embryo during gestation, is another stem cell source. Amnion-derived stem cells are classified as human amniotic membrane-derived epithelial stem cells, human amniotic membrane-derived mesenchymal stem cells and human amniotic fluid-derived stem cells. They are in an intermediate stage between pluripotent ESCs and lineage-restricted adult stem cells, non-tumorigenic, and contribute to low immunogenicity and anti-inflammation. Furthermore, they are easily available and do not cause any controversial issues in their recovery and applications. Not only are amnion-derived stem cells applicable in regenerative medicine, they have anticancer capacity. In non-engineered stem cells transplantation strategies, amnion-derived stem cells effectively target the tumor and suppressed the tumor growth by expressing cytotoxic cytokines. Additionally, they also have a potential as novel delivery vehicles transferring therapeutic genes to the cancer formation sites in gene-directed enzyme/prodrug combination therapy. Owing to their own advantageous properties, amnion-derived stem cells are emerging as a new candidate in anticancer therapy.