A case report of a family with developmental arrest of human prokaryotic stage zygote

To study the genetic variation leading to the arrest phenotype of pronuclear (PN) zygotes. We recruited a family characterized by recurrent PN arrest during in vitro fertilization (IVF) and intracytoplasmic sperm injection cycles (ICSI) and performed whole-exome sequencing for 2 individuals. The transcriptome profiles of PN-arrest zygotes were assessed by single-cell RNA sequencing analysis. The variants were then validated by PCR amplification and Sanger sequencing in the affected individuals and other family members. A family characterized by recurrent PN arrest during IVF and ICSI cycles were enrolled after giving written informed consent. Peripheral blood samples were taken for DNA extraction. Three PN-arrest zygotes from patient III-3 were used for single-cell RNA-seq as described. This phenotype was reproduced after multiple cycles of egg retrieval and after trying different fertilization methods and multiple ovulation regimens. The mutant genes of whole exon sequencing were screened and verified. The missense variant c. C1630T (p.R544W) in RGS12 was responsible for a phenotype characterized by paternal transmission. RGS12 controls Ca2+ oscillation, which is required for oocyte activation after fertilization. Single-cell transcriptome profiling of PN-arrest zygotes revealed defective established translation, RNA processing and cell cycle, which explained the failure of complete oocyte activation. Furthermore, we identified proximal genes involved in Ca2+ oscillation–cytostatic factor–anaphase-promoting complex (Ca2+ oscillation–CSF–APC) signaling, including upregulated CaMKII, ORAI1, CDC20, and CDH1 and downregulated EMI1 and BUB3. The findings indicate abnormal spontaneous Ca2+ oscillations leading to oocytes with prolonged low CSF level and high APC level, which resulted in defective nuclear envelope breakdown and DNA replication. We have identified an RGS12 variant as the potential cause of female infertility characterized by arrest at the PN stage during multiple IVF and ICSI

Human Papillomavirus Type 18 E6 and E7 Genes Integrate into Human Hepatoma Derived Cell Line Hep G2

Background and Objectives: Human papillomaviruses have been linked causally to some human cancers such as cervical carcinoma, but there is very little research addressing the effect of HPV infection on human liver cells. We chose the human hepatoma derived cell line Hep G2 to investigate whether HPV gene integration took place in liver cells as well. Methods: We applied PCR to detect the possible integration of HPV genes in Hep G2 cells. We also investigated the expression of the integrated E6 and E7 genes by using RT-PCR and Western blotting. Then, we silenced E6 and E7 expression and checked the cell proliferation and apoptosis in Hep G2 cells. Furthermore, we analyzed the potential genes involved in cell cycle and apoptosis regulatory pathways. Finally, we used in situ hybridization to detect HPV 16/18 in hepatocellular carcinoma samples. Results: Hep G2 cell line contains integrated HPV 18 DNA, leading to the expression of the E6 and E7 oncogenic proteins. Knockdown of the E7 and E6 genes expression reduced cell proliferation, caused the cell cycle arrest at the S phase, and increased apoptosis. The human cell cycle and apoptosis real-time PCR arrays analysis demonstrated E6 and E7-mediated regulation of some genes such as Cyclin H, UBA1, E2F4, p53, p107, FASLG, NOL3 and CASP14. HPV16/18 was found in only 9% (9/100) of patients with hepatocellular carcinoma. Conclusion: Our investigations showed that HPV 18 E6 and E7 genes can be integrated into the Hep G2, and we observed a low prevalence of HPV 16/18 in hepatocellular carcinoma samples. However, the precise risk of HPV as causative agent of hepatocellular carcinoma needs further study

Calculation of Bearing Capacity and Deformation of Composite Pile Foundation with Long and Short Piles in Loess Areas

In order to calculate the bearing capacity and settlement deformation of composite pile foundations with long and short piles in collapsible loess areas, the theoretical approximate solution was used to obtain the location of the neutral point of single piles. Additionally, based on the equation to calculate the bearing capacity of multielement composite foundations, a method considering the negative frictional resistance was proposed for calculating the bearing capacity of composite pile foundations with long and short piles. Based on the shear displacement method and the principle of deformation control, an equation to calculate the displacement and deformation of a composite pile foundation was presented. A model test with different operating conditions, i.e., a single pile, four piles, and eight piles, was designed to verify the proposed calculation methods. The results show that the location of the neutral point has a significant influence on the single-pile negative frictional resistance, and the neutral point ratio of the calculation meets the value range of the practical project. When the load at the top of the pile is relatively small, the experimental curve is consistent with the theoretical calculation curve, whereas when the load is comparatively large, the theoretically calculated displacement increase at the top of the pile is greater than the measured one. Under the premise that the theoretical calculation is in good agreement with the results, the theoretical value is larger than the actual value. And it contributes to strengthening engineering safety

Bearing Characteristics of Composite Pile Group Foundations with Long and Short Piles under Lateral Loading in Loess Areas

It is very necessary to research the bearing characteristics of composite pile group foundations with long and short piles under lateral load in loess areas, because these foundations are used widely. But few people researched this problem in loess areas up to now worldwide. In this paper, firstly, an indoor test model of a composite pile foundation with long and short piles is designed and then employed to explore the vertical load bearing characteristics and load transfer mechanisms of a single pile, a four-pile group, and a nine-pile group under different lateral loads. Secondly, ANSYS software is employed to analyze the load-bearing characteristics of the test model, and for comparison with the experimental results. The results demonstrate the following. (1) The lateral force versus pile head displacement curves of the pile foundation exhibit an obvious steep drop in section, which is a typical feature of piercing damage. A horizontal displacement limit of the pile foundation is 10 mm and 6mm for the ones sensitive to horizontal displacement. (2) The axial force along a pile and frictional resistance do not coincide, due to significant variations and discontinuities in the collapsibility of loess; a pile body exhibits multiple neutral points. Therefore, composite pile groups including both long and short piles could potentially maximize the bearing capacity and reduce pile settlement. (3) The distribution of stress and strain along the pile length is mainly concentrated from the pile head to a depth of about 1/3 of the pile length. If the lateral load is too large, short piles undergo rotation about their longitudinal axis and long piles undergo flexural deformation. Therefore, the lateral bearing capacity mainly relies on the strength of the soil at the interface with the pile or the horizontal displacement of the pile head

Silencing of Tumor Necrosis Factor Receptor 1 by siRNA in EC109 Cells Affects Cell Proliferation and Apoptosis

Tumor necrosis factor receptor 1 (TNFR1) is a membrane receptor able to bind TNF-α or TNF-β. TNFR1 can suppress apoptosis by activating the NF-κB or JNK/SAPK signal transduction pathway, or it can induce apoptosis through a series of caspase cascade reactions; the particular effect may depend on the cell line. In the present study, we first showed that TNFR1 is expressed at both the gene and protein levels in the esophageal carcinoma cell line EC109. Then, by applying a specific siRNA, we silenced the expression of TNFR1; this resulted in a significant time-dependent promotion of cell proliferation and downregulation of the apoptotic rate. These results suggest that TNFR1 is strongly expressed in the EC109 cell line and that it may play an apoptosis-mediating role, which may be suppressed by highly activated NF-κB

Transfection with E7-siRNA induced apoptosis.

<p>(A, B, C and D) The percent of apoptotic Hep G2 cells was measured using the Annexin V assay at 24 h, 48 h and 72 h after transfection, and then the stained cells were analyzed through flow cytometry. (E) The results showed that 7.26%±0.29%, 22.03%±0.23% and 19.20%±0.78% in siRNA E7 transfected Hep G2 cells after 24 h, 48 h and 72 h underwent total apoptosis compared with only 5.25%±0.76% in NC siRNA E7 transfected cells (p<0.05).</p

These photomicrographs show in situ hybridization results for human papillomavirus positive hepatocellular carcinoma.

<p>(A) Hep G2 cells were with the punctate signal pattern of HPV DNA. (B) HeLa cells were served as positive control. (C) Hepatocellular carcinoma was with diffuse signal pattern of HPV staining. (D) No signal was found in hepatoma carcinoma cells of this HPV- negative specimen.</p

Hep G2 cells with integrated HPV 18 DNA expressed E6 and E7 mRNAs and proteins.

<p>(A) PCR amplification of HPV 18 E6E7 gene was assessed in samples from Hep G2, EC109, and K562 cells. Then an amplified fragment of 847 bp was present in both Hep G2 and EC109 cells (HPV 18 positive), but absent in K562 cells (HPV 18 negative). (B) The expression of HPV 18 E6 and E7 mRNA was detected by RT-PCR. The expected fragments of E6 (196 bp) and E7 (332 bp) were present in both Hep G2 and EC109 cells, but not in K562 cells. (C) Western blotting showed the expression of the HPV 18 E6 and E7 proteins. EC109 and K562 were used as controls. β-actin was used as an internal control.</p

mRNA expression of E7-related genes in Hep G2 cells after the silencing of HPV 18 E7 through RT² Profiler™ Human Cell Cycle PCR Array.

<p>In 84 E7-related genes, 19 genes mRNA transcript were altered markedly compared with the negative control. Standards of eligibility: folds up- or down-regulation >2.0 and <i>p</i><0.05; n = 3.</p