Mechanistic Characterization of De Novo Generation of Variable Number Tandem Repeats in Circular Plasmids during Site-Directed Mutagenesis and Optimization for Coding Gene Application.

Site-directed mutagenesis for creating point mutations, sometimes, gives rise to plasmids carrying variable number tandem repeats (VNTRs) locally, which are arbitrarily regarded as polymerase chain reaction (PCR) related artifacts. Here, the alternative end-joining mechanism is reported rather than PCR artifacts accounts largely for that VNTRs formation and expansion. During generating a point mutation on GPLD1 gene, an unexpected formation of VNTRs employing the 31 bp mutagenesis primers is observed as the repeat unit in the pcDNA3.1-GPLD1 plasmid. The 31 bp VNTRs are formed in 24.75% of the resulting clones with copy number varied from 2 to 13. All repeat units are aligned with the same orientation as GPLD1 gene. 43.54% of the repeat junctions harbor nucleotide mutations while the rest don't. Their demonstrated short primers spanning the 3' part of the mutagenesis primers are essential for initial creation of the 2-copy tandem repeats (TRs) in circular plasmids. The dimerization of mutagenesis primers by the alternative end-joining in a correct orientation is required for further expansion of the 2-copy TRs. Lastly, a half-double priming strategy is established, verified the findings and offered a simple method for VNTRs creation on coding genes in circular plasmids without junction mutations.

High-Level Secretion of Pregnancy Zone Protein Is a Novel Biomarker of DNA Damage-Induced Senescence and Promotes Spontaneous Senescence.

Identification of unique and specific biomarkers to better detect and quantify senescent cells remains challenging. By a global proteomic profiling of senescent human skin BJ fibroblasts induced by ionizing radiation (IR), the cellular level of pregnancy zone protein (PZP), a presumable pan-protease inhibitor never been linked to cellular senescence before, was found to be decreased by more than 10-fold, while the level of PZP in the conditioned medium was increased concomitantly. This observation was confirmed in a variety of senescent cells induced by IR or DNA-damaging drugs, indicating that high-level secretion of PZP is a novel senescence-associated secretory phenotype. RT-PCR examination verified that the transcription of the PZP gene is enhanced in various cells at senescence or upregulated following DNA damage treatment in a p53-independent manner. Moreover, pretreatment with late pregnancy serum containing a high level of PZP led to inhibition of doxorubicin-induced senescence in A549 cells, and depletion of PZP in the pregnancy serum could enhance such inhibition. Finally, the addition of immuno-precipitated PZP complexes into tissue culture attenuated the growth of A549 cells and promoted the spontaneous senescence. Therefore, we revealed that high-level secretion of PZP is a novel and unique feature associated with DNA damage-induced senescence, and secreted PZP is a positive regulator of cellular senescence, particularly during the late stage of gestation.

Endometrial stromal sarcoma extending to the pulmonary artery: A rare case report.

BACKGROUND: Endometrial stromal sarcoma (ESS) is a rare malignant mesenchymal tumor. Early in the disease, the findings on magnetic resonance imaging are similar to those of leiomyoma. When the lesion involves both vascular and cardiac tissue, it might be misdiagnosed as intravenous leiomyomatosis, which is not common in the clinic. CASE SUMMARY: We present the case of a 34-year-old female patient with tumor embolus, which extended from the right iliac vein and ovarian vein to the inferior vena cava (IVC), and then to the right atrium and right ventricle, and finally protruded into the pulmonary artery. The patient had undergone a hystero-myomectomy 7 years previously. Based on the findings of the imaging examinations, the diagnosis of intravenous leiomyomatosis was considered preoperatively. The patient then underwent complete resection of the endovascular and intracardiac tumor embolus. The postoperative pathology results confirmed metastatic ESS with endovascular and intracardiac involvement. The patient was discharged from hospital in good condition, and there was no sign of recurrence 5 mo after the operation. CONCLUSION: Extending from the iliac vein and ovarian vein to the IVC, this metastatic ESS invaded both vascular and cardiac tissues. For patients with ESS involving vascular and cardiac tissues, pathological examinations are essential for the differential diagnosis, such as intravenous leiomyomatosis. In addition, due to the high recurrence rate of ESS, long-term and close follow-up evaluation is necessary.

Occipital nodular fasciitis easily misdiagnosed as neoplastic lesions: A rare case report.

BACKGROUND: Nodular fasciitis (NF) is a benign disease originating from fascial tissue and most commonly occurs in the extremities, followed by the trunk, head, and neck. NF of the head and neck occurs mainly in the face and neck, and it has not been reported in the occipital region. CASE SUMMARY: A 30-year-old man was admitted because of a mass in the left occipital region. Imaging examination revealed a soft tissue nodule in the left occipital area. An enhanced magnetic resonance imaging scan showed characteristic inverted target and fascial tail signs. Histopathological analysis showed a large amount of spindle cell proliferation, and immunohistochemistry showed positive expression of SMA in the spindle cells in the lesion. Finally, nodular fasciitis was diagnosed. CONCLUSION: NF of the head and neck is rare, but the possibility of NF should be considered when nodules or masses with rapid subcutaneous growth are found and tenderness in the head and neck is present. Imaging examination, in combination with clinical manifestations and histopathological examination, can improve the diagnostic accuracy for the disease. After diagnosis, local surgical resection is the first choice of treatment.

Loss of tumor suppressor inositol polyphosphate 4-phosphatase type B impairs DNA double-strand break repair by destabilization of DNA tethering protein Rad50.

Genome instability is the fundamental hallmark of malignant tumors. Tumor suppressors often play a role in maintaining genome stability. Our previous genetic screen identified inositol polyphosphate 4-phosphatase type B (INPP4B), primarily hydrolyzing phosphatidylinositol 3, 4-disphosphate, is a potential tumor suppressor in lung cancer cells. How INPP4B regulates the genome stability of lung cancer cells is unclear. Here we report knockout of INPP4B in lung adenocarcinoma A549 cells by Crispr-Cas9 gene editing leads to sensitization to ionizing radiation (IR), PARP inhibitor olaparib and impaired DNA homologous recombination repair. Re-introduction of a Crispr-Cas9 resistant INPP4B gene in the INPP4B knockout cells partially restored their resistance to IR, indicating loss of INPP4B protein is relevant to the increased IR sensitivity. Furthermore, we showed ectopic expressed INPP4B in A549 cells responds to IR irradiation by redistribution from cytoplasm to nucleus and endogenous INPP4B protein interacts with Rad50, a crucial MRN complex component for tethering DNA double-strand breaks. Loss of INPP4B protein results in decreased stability of Rad50 in vivo, suggesting an unanticipated role of tumor suppressor INPP4B in maintaining genome integrity via facilitating Rad50 mediated DNA double-strand break repair. Taken together, our findings support a dual role of INPP4B in suppression of tumorigenesis by safeguarding genome stability, as well as inhibiting of PI3K-Akt-mTOR signaling, and offer a new therapeutic strategy for personalized cancer treatment to patients with INPP4B defects or deficiency in the clinic.

A germline mutation in Rab43 gene identified from a cancer family predisposes to a hereditary liver-colon cancer syndrome.

BACKGROUND: Hereditary cancer syndromes have inherited germline mutations which predispose to benign and malignant tumors. Understanding of the molecular causes in hereditary cancer syndromes has advanced cancer treatment and prevention. However, the causal genes of many hereditary cancer syndromes remain unknown due to their rare frequency of mutation. METHODS: A large Chinese family with a history of hereditary liver-colon cancer syndrome was studied. The genomic DNA was extracted from the blood samples of involved family members, whole-exome sequencing was performed to identify genetic variants. Functional validation of a candidate variant was carried out using gene expression, gene knockout and immunohistochemistry. RESULTS: The whole-exome of the proband diagnosed with colon cancer was sequenced in comparison with his mother. A total of 13 SNVs and 16 InDels were identified. Among these variants, we focused on a mutation of Rab43 gene, a GTPase family member involving in protein trafficking, for further validation. Sanger DNA sequencing confirmed a mutation (c: 128810106C > T, p: A158T) occurred in one allele of Rab43 gene from the proband, that heterozygous mutation also was verified in the genome of the proband's deceased father with liver cancer, but not in his healthy mother and sister. Ectopic expression of the Rab43 A158T mutant in Huh7 cells led to more enhanced cell growth, proliferation and migration compared to the expression of wild type Rab43. Conversely, knockout of Rab43 in HepG2 cells resulted in slow cell growth and multiple nuclei formation and impaired activation of Akt. Finally, a positive correlation between the expression levels of Rab43 protein and cancer development in that family was confirmed. CONCLUSIONS: A germline mutation of Rab43 gene is identified to be associated with the onset of a familial liver-colon cancer syndrome. Our finding points to a potential role of protein trafficking in the tumorigenesis of the familial cancer syndrome, and helps the genetic counseling to the affected family members.