Identification of a metabolism-linked genomic signature for prognosis and immunotherapeutic efficiency in metastatic skin cutaneous melanoma.

Metastatic skin cutaneous melanoma (MSCM) is the most rapidly progressing/invasive skin-based malignancy, with median survival rates of about 12 months. It appears that metabolic disorders accelerate disease progression. However, correlations between metabolism-linked genes (MRGs) and prognosis in MSCM are unclear, and potential mechanisms explaining the correlation are unknown. The Cancer Genome Atlas (TCGA) was utilized as a training set to develop a genomic signature based on the differentially expressed MRGs (DE-MRGs) between primary skin cutaneous melanoma (PSCM) and MSCM. The Gene Expression Omnibus (GEO) was utilized as a validation set to verify the effectiveness of genomic signature. In addition, a nomogram was established to predict overall survival based on genomic signature and other clinic-based characteristics. Moreover, this study investigated the correlations between genomic signature and tumor micro-environment (TME). This study established a genomic signature consisting of 3 genes (CD38, DHRS3, and TYRP1) and classified MSCM patients into low and high-risk cohorts based on the median risk scores of MSCM cases. It was discovered that cases in the high-risk cohort had significantly lower survival than cases in the low-risk cohort across all sets. Furthermore, a nomogram containing this genomic signature and clinic-based parameters was developed and demonstrated high efficiency in predicting MSCM case survival times. Interestingly, Gene Set Variation Analysis results indicated that the genomic signature was involved in immune-related physiological processes. In addition, this study discovered that risk scoring was negatively correlated with immune-based cellular infiltrations in the TME and critical immune-based checkpoint expression profiles, indicating that favorable prognosis may be influenced in part by immunologically protective micro-environments. A novel 3-genomic signature was found to be reliable for predicting MSCM outcomes and may facilitate personalized immunotherapy.

C18H17NO6 Inhibits Invasion and Migration of Human MNNG Osteosarcoma Cells via the PI3K/AKT Signaling Pathway.

BACKGROUND Osteosarcoma (OS) is a highly aggressive, metastatic bone tumor with a poor prognosis, and occurs more commonly in children and adolescents. Therefore, new drugs and treatments are urgently needed. In this study, we investigated the effect and potential mechanisms of C18H17NO6 on osteosarcoma cells. MATERIAL AND METHODS Human MNNG osteosarcoma cells were treated with different concentrations of C18H17NO6. The proliferation of the MNNG cells was examined via CCK-8 assay. Cell migration and invasion were tested via wound-healing assay and Transwell migration and invasion assays. ELISA was used to detect MMP-2, MMP-9, and VEGF secretion. Finally, Western blotting and qRT-PCR were used to detect protein and mRNA expressions, respectively. RESULTS C18H17NO6 inhibited MNNG proliferation in a dose- and time-dependent manner and inhibited MMP-2, MMP-9, and VEGF secretion. C18H17NO6 treatment significantly downregulated N-cadherin and Vimentin expression levels and upregulated E-cadherin expression levels in vitro and in vivo. C18H17NO6 inhibited tumor growth in a MNNG xenograft. We also found that C18H17NO6 can significantly reduce the phosphorylation of the PI3K/AKT signaling pathway in vivo and in vitro. However, 740Y-P (a PI3K agonist) had the opposite effect on proliferation, migration and invasion of MNNG cells treated with C18H17NO6. LY294002 (a PI3K inhibitor) downregulated p-PI3K and p-AKT could mimic the inhibitory effect of C18H17NO6. CONCLUSIONS Our results suggest that C18H17NO6 can inhibit human MNNG osteosarcoma cell invasion and migration via the PI3K/AKT signaling pathway both in vivo and in vitro. C18H17NO6 may be a highly effective and low-toxicity natural drug for the prevention or treatment of OS.

A systematic review and meta-analysis of randomized placebo-controlled trials of DHEA supplementation of bone mineral density in healthy adults.

To find out if DHEA replacement improves bone mineral density (BMD) in healthy older adults. We systematically searched Medline via PubMed, Embase, and the Cochrane Library Center Register to identify randomized controlled trials up to October 2018. Effect estimates were performed in random effect models. Bone mineral density of hip and trochanter, total body, lumbar spine, and femoral neck were conducted. Hip BMD increased significantly above placebo group in women who took DHEA supplementation (SMD -0.5[-0.95, -0.04], p = .03). The SMD of trochanter BMD of women in placebo group than DHEA group was -0.55 [-1.10, 0.00], p = .05. Insulin-like growth factor 1 (IGF-1) did not change in men compared to placebo group also (-0.56 [-1.22, 0.10], p = .09). In women, IGF-1 significantly improved in DHEA supplementation group than placebo group (-2.61 [-4.85, -0.38], p = .02). In summary, the results of this meta-analysis suggest that DHEA replacement therapy can partially increase BMD of hip and trochanter in women. Similar results were not observed in men. More trials may be necessary to allow for a positive and clinically significant effect of DHEA on BMD.

Effect of abnormal GpG methylation in the second trimester of pregnancy on adverse health risk of offspring.

Effect of abnormal GpG methylation in amniotic fluid cells during the second trimester of pregnancy on adverse health risk of offspring was investigated. In total, 237 sets of amniotic fluid cells were collected from patients who received prenatal diagnosis in the Third Affiliated Hospital of Guangzhou Medical University (Guangzhou, China) from April 2010 to October 2011. Among them, 156 sets were from singleton and 81 sets were from twins. H19 gene was amplified by PCR, and the product was purified and pyrosequencing was used to detect the DNA methylation level of GapG. Follow-up records of the birth outcomes of pregnant women's offspring were collected. Positive rate of DNA amplification in 200 cases of amniotic fluid cells was 84.4% (200/237). Average age of singleton pregnancies was higher than that of twins (P<0.05), and no significant differences were found in gestational age and PCR amplification rate (P>0.05). There was no difference in the methylation level of GapG between singleton and twins (P>0.05), but the abnormal methylation rate of GapG1 in twin fetuses was significantly higher than that of singleton (20.3 vs. 3.6%, χ2=8.364, P=0.004). Offspring sex, singleton or twins, mode of delivery, time of pregnancy, and low birth weight showed no significant effect on GapG methylation level of H19 in the second trimester of pregnancy. No offspring deformities were found regardless of the increased or decreased degree of methylation (P>0.05). The number of fetuses born may cause abnormal GapG1 methylation, but no effect of GapG methylation on the adverse health risk of offspring was found.

Preparation and Characterization of Biochars from Eichornia crassipes for Cadmium Removal in Aqueous Solutions.

The study investigated the preparation and characterization of biochars from water hyacinth at 300°C to 700°C for cadmium (Cd) removal from aqueous solutions. The adsorption process was dominated by oxygen-containing functional groups with irregular surfaces via esterification reactions. Furthermore, the mineral components in the biochars also contributed to Cd absorption through precipitation. Parameters such as the effects of solution pH, contact time, and initial concentration were studied. The optimum pH value was observed at 5.0, in which nearly 90% of Cd was removed. The maximum Cd adsorption capacities based on the Langmuir isotherm were calculated at 49.837, 36.899, and 25.826 mg g(-1). The adsorption processes of the biochars followed the pseudo-second-order kinetics, with the equilibrium achieved around 5 h. The biochar from E. crassipes is a promising adsorbent for the treatment of wastewater, which can in turn convert one environmental problem to a new cleaning Technology.

Ecological risks assessment and pollution source identification of trace elements in contaminated sediments from the Pearl River Delta, China.

Sediments from 14 stations in the Foshan Waterway, a river crossing the industrial district of Guangdong Province, South China, were sampled and subsequently analyzed. The 14 stations were selected for the pollution discharging features of the river, such as the hydrology, the distribution of pollution sources, and the locations of wastewater outlets. The ecological risks were assessed, and the pollution sources were identified to provide valuable information for environmental impact assessment and pollution control. The spatial variability was high and the range were (in milligrams per kilogram dry weight): Pb, 46.0~382.8; Cu, 33.7~ 482.3; Zn, 62.2~1,568.7; Ni, 28.5~130.7; Cr, 34.7~1,656.1; Cd, 0.50~8.53; Hg, 0.02~8.27; and As, 5.77~66.09. The evaluation results of enrichment factor and potential ecological risk index indicate that the metal pollution in the surface and bottom sediments were severely polluted and could pose serious threat to the ecosystem in most stations. Although the hazard levels of the trace element differed among the stations, Hg was the most serious pollutant in all stations. The results of principal component analysis (PCA) show that the discharge of industrial wastewater is the most important polluting factor whereas domestic sewage, which contains a large amount of organic substances, accelerates metal deposition. And potential pollution sources were identified by the way of integrating the analysis results of PCA and data gained from the local government. Therefore, the conclusion is drawn that Foshan Waterway is seriously polluted with trace elements, both in the surface sediment (0 to 20 cm) and the bottom sediments (21 to 50 cm) are contaminated.

Single-nucleotide polymorphism array coupled with multiple displacement amplification: accuracy and spatial resolution for analysis of chromosome copy numbers in few cells.

When coupled with multiple displacement amplification (MDA), microarray-based comparative genomic intensity allows detection of chromosome copy number aberrations even in single or few cells, but the actual performance of the system and their influencing factors have not been well defined. Here, using single-nucleotide polymorphism (SNP) array, we analyzed copy number profiles from DNA amplified by MDA in 1-10 cells and estimated the accuracy and spatial resolution of the analysis. Based on the concordance of SNP copy numbers for DNA with and without MDA, the accuracy of the system can be significantly enhanced by using MDA-amplified DNA as reference and also by increasing the cell numbers. Analyses under different smoothing treatments revealed a practical resolution of 2 Mb for 10 cells and 10 Mb for a single cell. When both cells with known chromosomal duplication and deletion were analyzed, this platform detected a copy number "loss" more accurately than a "gain" (P < 0.01), particularly in single-cell MDA products. Together, we demonstrated that SNP array coupled with MDA is reliable and efficient for detection of copy number aberrations in a small number of cells, and its accuracy and resolution can both be significantly enhanced with increasing the number of cells as MDA template.

Derivation of cloned human blastocysts by histone deacetylase inhibitor treatment after somatic cell nuclear transfer with ß-thalassemia fibroblasts.

Derivation of embryonic stem cells from patient-specific cloned blastocysts by somatic cell nuclear transfer (SCNT) holds promise for both regenerative medicine and cell-based drug discovery. However, the efficiency of blastocyst formation after human SCNT is very low. The developmental competence of SCNT embryos has been previously demonstrated in several species to be enhanced by treatment with histone deacetylase inhibitors, such as trichostatin A (TSA), to increase histone acetylation. In this study, we report that treatment of SCNT embryos with 5 nM TSA for 10 h following activation incubation increased the developmental competence of human SCNT embryos constructed from ß-thalassemia fibroblast cells. The efficiency of blastocyst formation from SCNT human embryos treated with TSA was approximately 2 times greater than that from untreated embryos. Cloned blastocysts were confirmed to be generated through SCNT by DNA and mitochondrial DNA fingerprinting analyses. Further, treatment of SCNT embryos with TSA improved the acetylation of histone H3 at lysine 9 in a manner similar to that observed in in vitro fertilized embryos.

[Development and genetic polymorphism of abnormal pronuclear zygotes after intracytoplasmic sperm injection].

OBJECTIVE: To compare the development of abnormal pronuclear zygotes after intracytoplasmic sperm injection (ICSI) and analyze their genetic polymorphism. METHODS: Four hundred and ninety three abnormal pronuclear zygotes after ICSI were divided into three groups based on the number of pronuclei: 347 nonpronuclear oocytes, 71 monopronuclear zygotes and 75 multipronuclear zygotes. All of them were cultured in the medium of Vitrolife G5 series(TM). Sixteen short tandem repeats (STR) of seven blastocysts were then analyzed by ABI3100. RESULTS: The cleavage rate of nonpronuclear group (25.4%) was lower than that of the others (P<0.01), the proportion of blocked embryos in nonpronuclear group (48.9%) was significantly higher than that of the others (P<0.05), but the blastocyst rate showed no significant difference in three groups (P>0.05). The genetic polymorphism of the 16 STRs showed that the blastocysts from the nonpronuclear and multipronuclear were diploid, and one of the blastocysts from nonpronuclear oocyte was Y-bearing. CONCLUSION: The zygotes with abnormal pronuclei after ICSI might have development potential, and the blastocysts from nonpronuclear oocytes and multipronuclear zygotes could be diploid.

Monozygotic quadruplets after in vitro fertilization and embryo transfer.

A unique case is reported of a quadruple gestation (monochorionic quadramniotic quadruplets) after in vitro fertilization (IVF) and the transfer of two embryos. On the ninth week of pregnancy, a transvaginal sonogram revealed that there was no heart beat in any of the fetuses. Uterine curettage was then performed. The fetal karyotype was 46, XX, inv (9) (p11q13).

Similar DNA methylation and histone H3 lysine 9 dimethylation patterns in tripronuclear and corrected bipronuclear human zygotes.

After fertilization, male and female gametes undergo extensive reprogramming to restore totipotency. Both DNA methylation and histone modification are important epigenetic reprogramming events. Previous studies have reported that the paternal pronucleus of the human zygote is actively demethylated to some extent, while the maternal pronucleus remains methylated. However, to our knowledge, the relationship between DNA methylation and H3K9 dimethylation patterns in human embryos has not been reported. In this study, we examined the dynamic DNA methylation and H3K9 dimethylation patterns in triploid and bipronucleated zygotes and early developing embryos. We sought to gain further insight into the relationship between DNA methylation and H3K9 dimethylation and to investigate whether removing a pronucleus from triploid zygotes affects DNA methylation and H3K9 dimethylation patterns. We found that active DNA demethylation of the two male pronuclei occurred in tripronuclear human zygotes while the female pronucleus remained methylated at 20 h post-insemination. In tripronuclear human zygotes, H3K9 was hypomethylated in the two paternal pronuclei relative to the maternal pronucleus. Our data show that there are no differences in the DNA methylation and H3K9 dimethylation patterns between tripronuclear and corrected bipronuclear human zygotes. However, correction of 3PN human zygotes dispermic in origin could not improve subsequent embryo development. In conclusion, DNA methylation and H3K9 dimethylation patterns are well correlated in tripronuclear zygotes and embryos; early embryo development is not affected by removal of a male pronucleus. Our results imply that limited developmental potential of either 3PN or corrected 2PN embryos may not be caused by the abnormalities in DNA methylation or H3K9 dimethylation modification.

[In vitro maturation of human oocytes from pregnant late term: a new source of oocytes].

OBJECTIVE: To investigate the in vitro maturation of human oocytes (IVM) from pregnant late term, natural cycles and Gn stimulating cycles and the effect of granulose cells on IVM from pregnant late term. METHODS: A total of 1086 immature oocytes were obtained including 633 oocyte-cumulus complexes (OCCs) and 453 denuded oocytes (DOs). OCCs were divided into pregnant late term group, natural cycle group and IVM group, and DOs were divided into pregnant late term group, natural cycle group and controlled ovarian hyperstimulation (COH) group. All the oocytes were matured in IVM culture system and fertilized by ICSI. The embryos were cultured to blastocyst stage except that those in IVM group were transferred into the uterus. The main outcomes were assessed including maturation rate (MR), fertilization rate (FR), cleavage rate (CR), and blastulation rate (BR) (natural cycle group, pregnant late term group and COH group) and pregnancy rate per transfer cycle (PR) of IVM group. RESULTS: MR of OCCs in pregnant late term group, natural cycle group and COH group was 74.3%, 76.9% and 82.2%, respectively, showing statistical difference between pregnant late term cycle group and IVM group. No statistical difference was observed in FR, CR or BR between the three groups. For IVM cycle group, clinical pregnancy rate of 20% per aspiration was achieved. For DOs, MR of COH group (86.0%) was significantly higher than that of the natural cycle group (72.5%) and pregnant late term group (72.7%) (P<0.01). FR, CR and BR showed no statistical difference among the 3 groups. No difference was found in MR, FR, CR and BR between OCCs group and DOs group from pregnant late term. CONCLUSIONS: The oocytes from pregnant late term have the same development potential as those from natural cycles or Gn stimulating cycles in vitro, and provide a new source of donor oocytes. Granulose cells do not affect the IVM from pregnant late term.

Derivation and characterization of human embryonic stem cell lines from poor quality embryos.

Poor quality embryos discarded from in vitro fertilization (IVF) laboratories are good sources for deriving human embryonic stem cell (hESC) lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses (ICMs) could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods (P>0.05). As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.

[Sperm acrosin activity helps predict IVF-ET outcome].

OBJECTIVE: To investigate the effect of sperm acrosin activity on the IVF-ET outcome. METHODS: We analyzed sperm parameters, morphology and acrosin activity for 909 infertile husbands by computer-assisted self-assessment (CASA), modified Papanicolaou staining and N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA), respectively, and detected the rates of fertilization, cleavage, quality embryos, embryo cryopreservation, implantation, clinical pregnancy and abortion. The wives were identified as normal or with mere oviduct problems. RESULTS: The rate of normal sperm morphology and sperm motility, vitality, rapid progressive velocity and concentration were significantly lower in the abnormal acrosin activity group than in the normal one (P < 0.01). Significant positive correlations were observed between acrosin activity and the above-mentioned semen parameters (P < 0.01). There were no significant differences in the number of retrieved eggs, the rates of cleavage, quality embryos, embryo cryopreservation, non-embryo transfer cycles and miscarriages, and the number of transferred embryos between the two groups (P > 0.05). The fertilization rate, the percentage of transfer cycles with only 1 embryo and the rate of implantation and clinical pregnancy were notably higher in the normal acrosin activity group than in the abnormal one (P < 0.01). CONCLUSION: Sperm acrosin activity is closely related with semen parameters, and it helps to predict the sperm fertilizing capacity and IVF-ET outcome.

Similar biological characteristics of human embryonic stem cell lines with normal and abnormal karyotypes.

BACKGROUND: Human embryonic stem cell (hESC) lines derived from poor quality embryos usually have either normal or abnormal karyotypes. However, it is still unclear whether their biological characteristics are similar. METHODS: Seven new hESC lines were established using discarded embryos. Five cell lines had normal karyotype, one was with an unbalanced Robertsonian translocation and one had a triploid karyotype. Their biological characteristics, short tandem repeat loci, HLA typing, differentiation capability and imprinted gene, DNA methylation and X chromosome inactivation status were compared between different cell lines. RESULTS: All seven hESC lines had similar biological characteristics regardless of karyotype (five normal and two abnormal), such as expression of stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-81 and TRA-1-60 proteins, transcription factor octamer binding protein 4 mRNA, no detectable expression of SSEA-1 protein and high levels of alkaline phosphatase activity. All cell lines were able to undergo differentiation. Imprinted gene expression and DNA methylation were also similar among these cell lines. Non-random X chromosome inactivation patterns were found in XX cell lines. CONCLUSIONS: The present results suggest that hESC lines with abnormal karyotype are also useful experimental materials for cell therapy, developmental biology and genetic research.